Genetically engineered Vibrio sp. and uses thereof

ABSTRACT

The present disclosure provides engineered Vibrio sp. organisms. The organisms can be engineered to have an altered Chromosome II so that the altered Chromosome II can be used in Vibrio sp. and other organisms for the cloning or amplification of nucleic acid molecules and for the expression and production of proteins and peptides in a Vibrio sp. organism. One or more genetic elements have been deleted from Chromosome II and/or relocated from Chromosome II to Chromosome I. The engineered Vibrio sp. organisms of the invention can also have signal sequences fused to proteins or peptides to be secreted from the cell. In some embodiments the engineered Vibrio sp. organisms can have sequences that enable them to retain viability after incubation at low temperatures.

This application claims the benefit of U.S. provisional application Ser. No. 62/380,341, filed Aug. 26, 2016, which is incorporated by reference herein it its entirety, including all tables, figures, and claims.

INCORPORATION OF SEQUENCE LISTING

The material in the accompanying sequence listing is hereby incorporated by reference into this application. The accompanying sequence listing text file, name SGI2050_1_Sequence_ST25, was created on Aug. 26, 2016, and is 42,121 bytes. The file can be accessed using Microsoft Word on a computer that uses Windows OS.

FIELD OF THE INVENTION

The present disclosure relates to genetically engineered Vibrio sp. bacteria and the use of such bacteria for the cloning, construction, maintenance, manipulation and/or propagation of DNA molecules and for the expression and harvesting of protein and peptide sequences.

BACKGROUND OF THE INVENTION

The biotechnology sector relies upon organisms such as E. coli as hosts for the generation of desired biomolecules (e.g., recombinant DNA, proteins, natural products, etc.) as well as for the study of biological processes and the development of bio-based technologies and products. While advances in fields such as genomics, synthetic biology, and genome/metabolic engineering have made possible projects at an unprecedented scale, the host organisms that the field relies upon have changed relatively little in decades and are proving to be inadequate or inefficient for many ambitious projects.

E. coli has been the main prokaryotic workhorse for several decades, being used ubiquitously in both academic and industrial efforts, and relied upon as a host for molecular cloning, protein expression, metabolic engineering, a source of cellular extracts for in vitro molecular biology, and as a chassis for synthetic biology efforts. The use of E. coli is due largely to its extensive characterization (having served as a model organism since the late 19th century), having a large collection of standardized tools and protocols, and being relatively easy to work with. E. coli is certainly not the only organism in use in biotechnology, as there are plenty of obscure organisms being utilized, usually to leverage some peculiar biological property that allows that organism to excel in some niche application, but E. coli is the most widely adopted and broadly applied bacterial species in biotechnology.

There is a need for more robust, faster growing, and easily genetically manipulated bacterial cells that can be used as host organisms, especially to produce products such as large recombinant DNA molecules, and as alternative hosts for protein expression.

SUMMARY OF THE INVENTION

The present disclosure provides engineered Vibrio sp. organisms. The organisms can have an altered Chromosome II that serves as a vector within Vibrio and other organisms, or for the cloning of nucleic acid molecules and for the expression and production of proteins and peptides in a Vibrio sp. organism. Chromosome I and/or Chromosome II can also be engineered to contain one or more genetic elements for the cloning and amplification of the chromosome. The genetic elements can enable the cell to express an exogenous or heterologous protein or peptide, which can also be secreted from the cell. The engineered organisms of the invention can also contain a nucleic acid construct comprising a signal sequence, which enables the cell to secrete an expressed protein or peptide. In some embodiments the organisms of the invention have been engineered to survive, propagate, and amplify at cold temperatures. The genetically engineered organisms are useful for the construction, maintenance, manipulation, and/or propagation of DNA constructs; protein or peptide expression and/or secretion; metabolic engineering; expression of cellular extracts for cell-free biology; and for synthetic biology applications. The disclosure also relates to the use of the replication machinery of Vibrio sp. on a cloning vector for cloning or replication of recombinant DNA constructs or for the expression and production of heterologous protein and peptides by the Vibrio sp. cell.

In a first aspect the present invention provides a Vibrio sp. organism having a Chromosome I (ChI) comprising essential genetic elements and an altered Chromosome II (ChII) comprising at least one piece of exogenous DNA of greater than 10 kb in size that encodes at least one heterologous protein or peptide; and wherein the organism comprises all cellular components functional for the replication and amplification of the altered Chromosome II, and the altered Chromosome II is replicated during cellular growth of the organism. In one embodiment ChII of the organism comprises SEQ ID NO: 1 or a variant thereof. The at least one piece of heterologous DNA comprised on the altered ChII can be greater than 50 kb in size or greater than 100 kb or greater than 500 kb or greater than 1 Mb or greater than 2 Mb.

In some embodiments the altered ChII further encodes at least one origin of replication and at least one selection marker. The organism can have tetA/tetR or chloramphenicol resistance genes, an R6Kgamma origin of replication, and can also have an RP4 oriT region. In some embodiments at least one essential genetic element removed from ChII is comprised on ChI. The organism can be a Vibrio sp., which can be Vibrio natriegens.

In some embodiments the altered ChII has an inducible promoter, which optionally can be the IPTG inducible trc promoter, or the temperature inducible lambda pR promoter, or the arabinose inducible araBAD promoter.

In some embodiments ChI or ChII can have at least one deletion of a gene encoding a protein such as, for example, a recombinase, an endonuclease, a protease, and a restriction enzyme. The recombinase can be recA and the endonuclease can be Dns.

In another aspect the invention provides an organism having a DNA sequence that encodes a functional signal peptide operably linked to at least one heterologous protein or peptide. The heterologous protein or peptide can be expressed having the functional signal peptide so that the exogenous protein or peptide is secreted from the organism. The functional signal peptide can be any of SEQ ID Nos: 8-28 or a variant of any of them. The DNA sequence encoding the functional signal peptide operably linked to at least one exogenous protein or peptide can be comprised on a vector. The organism can be a Vibrio sp. organism.

In another aspect the invention provides a Vibrio sp. organism having an exogenous sequence encoding one or more enzymes from a reactive oxygen species detoxification system. In various embodiments the organism can have one or more alkyl hydroperoxide reductase gene(s) under the control of an exogenous promoter and operable in Vibrio sp., or a catalase gene under the control of an exogenous promoter and operable in Vibrio sp., or a glutathione S-transferase gene under the control of an exogenous promoter and operable in Vibrio sp., or any combination of them. In one embodiment the organism has the alkyl hydroperoxide reductase operon, which can be from E. coli (ahpCF). In another embodiment the organism has the catalase gene, which can be katG or katE. When the organism has the glutathione S-transferase gene it can be, in one embodiment, gstA. The reactive oxygen species detoxification system can be comprised on ChI or ChII. In some embodiments the organism can have an exogenous vector, and enzyme from the reactive oxygen species detoxification system can be comprised on the exogenous vector. In one embodiment the organism is comprised in a controlled environment at a temperature of about 4° C. or less. The organism can be comprised on a solid media. The organism having the enzyme from the reactive oxygen detoxification system can remain viable after cultivation at about 4° C. for at least 14 days, or can remain viable after cultivation at about 4° C. for at least 19 days.

In another aspect the invention provides a nucleic acid expression cassette encoding a. an inducible promoter operable in Vibrio sp.; b. a signal peptide functional to cause the secretion of an exogenous protein or peptide from the Vibrio sp. cell; c. an heterologous nucleic acid sequence; wherein the promoter, signal peptide, and heterologous nucleic acid sequence are operably linked for the expression and secretion of a protein or peptide encoded by the heterologous nucleic acid sequence from the Vibrio sp. cell. The promoter can be an inducible promoter, e.g. inducible with sucrose. In various embodiments the signal peptide can be any disclosed herein.

In another aspect the invention provides a nucleic acid shuttle vector containing a) a Vibrio sp. sequence of Chromosome II replication machinery; b) an origin of replication active in Vibrio sp.; c) an origin of replication active in a non-Vibrio organism; d) a selection marker; e) a gene of interest. In some embodiments the non-Vibrio organism is E. coli.

In another aspect the invention provides a Vibrio sp. organism containing a. a Chromosome I comprising a heterologous nucleic acid construct having a sequence for an inducible RNA polymerase, b. where the RNA polymerase is functionally active upon induction with an activator to activate a promoter operably linked to a heterologous nucleic acid on an extra-chromosomal DNA; and c. where the organism is a competent organism. The organism can have a Dns deletion and optionally a selectable marker.

In another aspect the invention provides a method of transforming a Vibrio sp. cell by a. providing a competent Vibrio sp. organism having a Chromosome I that comprises a heterologous sequence encoding an inducible RNA polymerase; b. introducing an extra-chromosomal DNA comprising a promoter operably linked to a heterologous nucleic acid on an extra-chromosomal DNA, wherein the promoter is activated by the RNA polymerase of a). The method can further comprise inducing the RNA polymerase of a). In some embodiments the method involves expressing the heterologous nucleic acid on the extra-chromosomal DNA to produce at least one protein.

The use of sub-titles of section headings herein is for ease of understanding the content of the application only and the subject matter under any specific heading or sub-heading is not to be construed as limited only to that described by the heading or sub-heading unless specifically indicated.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-F show the use of V. natriegens as a host for inducible protein expression. Six plasmids were designed for inducible protein expression of GFP: A. pBR322-trc-GFP; B. p15a-trc-GFP; C. pBR322-araBAD-GFP; D. p15 a-araBAD-GFP; E. pBR322-cI857ts-GFP and F. pl5a-cI857ts-GFP.

FIG. 2 shows GFP fluorescence normalized to OD600 for induced and non-induced cultures harboring each of the six expression plasmids.

FIG. 3 shows the presence of GFP from cultures of V. natriegens harboring pBR322-trc-GFP. Lane 1: SeeBlue Plus2 Protein Standard; Lane 2: 1 μL V. natriegens pBR322-trc-GFP lysate from IPTG-induced culture; Lane 3: 10 μL V. natriegens pBR322-trc-GFP lysate from IPTG-induced culture; Lane 4: 1 μL V. natriegens pBR322-trc-GFP lysate from non-induced culture and Lane 5: 10 μL V. natriegens pBR322-trc-GFP lysate from non-induced culture. Arrows indicate GFP.

FIG. 4 shows a plasmid comprising the replication machinery sequence from V. natriegens chrII, the R6Kγ origin of replication and the tetA/tetR resistance genes along with the RP4 oriT region of plasmid pJB3Tc20, which facilitates mobilization of the plasmid by bacterial conjugation.

FIG. 5 shows plasmid pVnoriCII-Mgen25-49 run on an agarose gel.

FIG. 6 shows the vector map and sequence of pVnatCII-YACTRP-copy control.

FIG. 7 shows a region of V. natriegens strain CCUG16374 chromosome I containing a 28 kb genetic island demarcated by the black arrow. Genes are depicted with grey arrows. The enzymes involved in the putative restriction-modification system are demarcated by striped arrows.

FIG. 8 A-B shows V. natriegens cultures comprising T7 RNA polymerase operably linked to the indicated inducible promoter and a GFP cassette operably linked to a T7 promoter in the indicated media. A) White light image of cultures. Two left most cultures are wild type strains not expressing GFP, while the other four cultures have a distinct yellow/green color, indicating expression of GFP. B) Blue light transilluminator image displaying the positive expression of GFP in the right four cultures, while the two wild type cultures on the left are not expressing GFP and therefore lack the trademark green fluorescent color.

FIG. 9 shows a stained gel of bacterial lysates from wild type (two left cultures) and the indicated four strains expressing inducible GFP. The dark band demarcated with the asterisk is the GFP protein, which is only present in the engineered strains (BHI lacI, LB lacI, BHI araBAD, LB araBD), and not wild type (wt) cultures.

FIG. 10A-10C are schematic illustrations of plasmids conferring cold tolerance to Vibrio sp. organisms. The PCR product for the 1566 bp ahpCF operon begins at the ahpC start codon (ATG), includes the native ahpF promoter, and ends at the ahpF stop codon. ahpCF and katE were amplified from E. coli strain W3110 while katG was amplified from E. coli strain BW25141. The plasmids also contain an arabinose-inducible copy of katE (FIG. 10A), katG (FIG. 10B), or ahpCF (FIG. 10C), an origin of transfer from plasmid RK2 (aka RP4) for mobilization via conjugation, a tetracycline resistance cassette, and the p15A origin of replication.

FIG. 11 shows the 20 most abundant proteins identified in growth media containing sucrose from Vibrio natriegens.

FIG. 12 is a depiction of an SDS-PAGE gel showing the secretome of V. natriegens strains grown on different carbon sources. When 0.2% sucrose (w/v) was spiked into a minimal media containing 3% glucose (w/v), a large protein band appeared between 49 kDa and 62 kDa.

FIG. 13 is a comparison of plasmid DNA isolated from V. natriegens strain E with or without the endogenous Dns nuclease. Plasmid pACYC184 was isolated from strain E with or without a chromosomal deletion of Dns and purified using a commercial prep kit with or without a PB wash step. The isolated DNA was then subjected to restriction digest with NheI and analyzed by gel electrophoresis. Without the PB wash step (which removes nuclease) during plasmid isolation, the DNA is completed degraded by the Dns nuclease; however, in a nuclease deficient strain, the PB wash step was not required to preserve the integrity of the DNA.

FIG. 14A is an illustration of the viability of V. natriegens colonies on LB agar plates stored at 4° C. vs. room temperature. The Figure demonstrates that V. natriegens stored as colonies on LB agar plates remain viable when stored for 20 days at room temperature, but not at 4° C. A variant of V. natriegens CCUG 16374 was initially streaked out on LB agar plates and incubated at either room temperature (RT) or 4° C. for 20 days. After 20 days of storage, eight colonies each from the plates stored at 4° C. (top row) and room temperature (bottom row) were streaked out on quadrants of fresh LB agar plates and incubated overnight at 30° C. No growth was observed from the eight colonies re-streaked from plates stored at 4° C. for 20 days (top row). Robust growth was observed from the eight colonies re-streaked from plates stored at RT for 20 days (bottom row).

FIG. 14B demonstrates that catalase exhibits a protective effect on V. natriegens cells stored as colonies on agar plates at 4° C. A variant of V. natriegens CCUG 16374 was plated out onto an LB agar plate and incubated overnight at 30° C. This plate was then stored at 4° C. After 10 days at 4° C., a colony was streaked out on both BHI/v2 agar plates and LB agar plates with or without the addition of 2000 units of bovine catalase. The streak plates were incubated overnight at 30° C. and examined the following day. Note that plating on plates containing catalase dramatically increases the viability of cold-stored cells.

FIG. 14C demonstrates the improved tolerance to prolonged cold storage at 4° C. of a V. natriegens strain harboring plasmids encoding for E. coli ROS detoxification systems. LB+tetracycline (15 ug/mL) agar plates with colonies of a variant of V. natriegens (CCUG 16374) containing plasmids encoding the E. coli ahpCF operon, katE, or katG were stored at 4° C. for 21 days. On Day 11 and day 21, eight single colonies from each plate were picked using sterile inoculating loops and struck in quadrants on fresh LB+tetracycline (15 ug/mL) plates. The plates were incubated overnight and examined for bacterial growth. The presence of the plasmid-borne detox systems improved the viability of V. natriegens upon prolonged storage at 4° C., with katG having the most robust effect.

FIG. 15A-B. FIG. 15A illustrates two chromosomes of V. natriegens strain E depicted with regions not present in V. natriegens strain A annotated in light red. An enlargement of three regions present in V. natriegens strain E, but absent from V. natriegens strain A are shown in the dashed blue boxes. Coding sequences are colored in yellow, genes associated with insertion sequences are colored in blue, genes annotated as associated with restriction/modification systems colored in pink, phage-related proteins shown in purple, and genes showing high similarity to proteins in the REBASE restriction enzyme database shown in dark red. FIG. 15B provides a list of genes that can be deleted. The organism of the invention can contain a deletion of any one or more of the genes listed in Table 15B and any combination of them.

FIG. 16 provides an image of an SDS-PAGE gel of the supernatant V. natriegens comprising the signal sequence of SEQ ID NO: 8 fused to modified forms of the endogenous V. natriegens levansucrase as three recombinant constructs, C2, G2, and H2. The Figure shows all three constructs were efficiently secreted by V. natriegens. The molecular weight markers are (from bottom to top): 10, 15, 25, 30, 40, 50, 70, 80, 115, 140 kD.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides engineered Vibrio sp. organisms. In some embodiments the organisms comprise a vector and are capable of cloning large sequences of DNA of greater than 10 kb or greater than 50 kb in size, or larger. In other embodiments the invention provides signal sequences for use in Vibrio sp. and other organisms and can be utilized for the secretion of a heterologous protein of interest. The signal sequences can achieve high secretion levels of desired heterologous proteins. In other embodiments the invention provides engineered Vibrio sp. organisms that can tolerate low temperatures on solid or semi-solid media and sustain viability or culturability of the cells after being stored at the low temperatures. The invention allows for the acceleration of various applications in molecular biology, synthetic biology, and metabolic engineering due to the rapid growth rate nutritional versatility of the organism.

Vibrio is a genus of Gram-negative, facultative anaerobic bacteria possessing a curved-rod shape, with Vibrio sp. indicating a species within the genus Vibrio. In some embodiments, Vibrio sp. can comprise any one or more of the following Vibrio species, and in all possible combinations: adaptatus, aerogenes, aestivus, aestuarianus, agarivorans, albensis, alfacsensis, alginolyticus, anguillarum, areninigrae, artabrorum, atlanticus, atypicus, azureus, brasiliensis, bubulus, calviensis, campbellii, casei, chagasii, cholera, cincinnatiensis, coralliilyticus, crassostreae, cyclitrophicus, diabolicus, diazotrophicus, ezurae, fischeri, fluvialis, fortis, furnissii, gallicus, gazo genes, gigantis, halioticoli, harveyi, hepatarius, hippocampi, hispanicus, hollisae, ichthyoenteri, indicus, kanaloae, lentus, litoralis, logei, mediterranei, metschnikovii, mimicus, mytili, natriegens, navarrensis, neonates, neptunius, nereis, nigripulchritudo, ordalii, orientalis, pacinii, parahaemolyticus, pectenicida, penaeicida, pomeroyi, ponticus, proteolyticus, rotiferianus, ruber, rumoiensis, salmonicida, scophthalmi, splendidus, superstes, tapetis, tasmaniensis, tubiashii, vulnificus, wodanis, and xuii. In some embodiments, Vibrio sp. is Vibrio natriegens. In some embodiments, Vibrio sp. is not Vibrio cholera. In some embodiments, Vibrio sp. comprises all species of Vibrio other than cholera.

Vibrio sp. have several advantages as host cells over other bacteria for many molecular biology applications. One advantage is their rapid growth rate. One of the most time intensive steps in modern biotech workflows is waiting for the host cell to grow to a sufficient density before DNA/protein/product can be recovered or the phenotype can be assessed. Since dramatic time savings have been realized in other areas of biotech workflows (e.g., sequencing, bioinformatic analysis, high-throughput assays, etc.), growth of the host has become an even more significant bottleneck. E. coli is considered to have one of the quickest growth rates relative to other organisms used in the biotech sector, and this has been one of its strengths. Because Vibrio sp. have a growth rate 2-3× faster than commonly used E. coli strains, it is able to achieve a dramatic reduction in the time necessary for the host to grow, and will accelerate research efforts. In certain aspects of the present disclosure, the growth rate of Vibrio sp. in terms of doubling time expressed as numbers of cells in a population is about 10 minutes or about 12 minutes or about 13 minutes or about 14 minutes or about 15 minutes or about 16 minutes. In other aspects, the growth rate of a genetically engineered Vibrio sp. in terms of doubling time is 5-30 minutes.

Another advantage of Vibrio sp. is the size of heterologous DNA that can be harbored. Large scale genetic engineering or synthetic genome construction efforts require the assembly, manipulation, and maintenance of large pieces of recombinant DNA, tasks which are carried out in a genetically tractable host (such as E. coli) before delivery of the engineered DNA to the final host organism. Currently, most of this work is carried out in E. coli, but as projects become more ambitious, the limitations of this species are becoming apparent. It has been observed that with current technologies, E. coli is capable of harboring exogenous DNA constructs of no more than 500 kb (and in some cases much less depending on the properties of the DNA being cloned) on a bacterial artificial chromosome, which is a serious limitation for synthetic genome/large pathway construction efforts. This has necessitated the development of novel hosts as cloning platforms such as Saccharomyces cerevisiae and Bacillus subtilis. While these hosts have the advantage of being able to take up and stably propagate large (Mb-sized) fragments of heterologous DNA, they have their own disadvantages, with Saccharomyces cerevisiae growing much slower than E. coli (˜3× slower), and both species being incompatible with standard laboratory techniques and being very difficult to recover DNA from.

An additional advantage is the compatibility of Vibrio sp. with standard lab protocols. Unlike organisms that require specialized techniques or methods, Vibrio sp. is compatible with many standard cloning vectors, growth media, workflows and commercially-available kits developed for E. coli or recovering DNA.

A further advantage is the nutritional versatility of Vibrio sp. allowing it to grow on a range of different growth media, including inexpensive, minimal media. Coupled with its rapid growth rate, this feature allows for industrial scale production in less time and at lower cost.

Chromosomes I and II

The two chromosomes of the genus Vibrio are designated as Chromosome I (ChI) and Chromosome II (ChII). Chromosome I in the natural state is typically from 3.0-3.3 Mb and Chromosome II in the natural state from 0.8-2.4 Mb but the particular numbers depend on the species of Vibrio. Some embodiments of the invention comprise a Vibrio sp. having an altered Chromosome I and/or an altered Chromosome II.

In one embodiment the Vibrio host cell of the invention has an altered Chromosome II that has had non-essential genetic elements deleted (and which can be relocated to Chromosome I or extrachromosomal DNA), thus freeing Chromosome II for use as a vector. There can be a copy of each essential genetic element on at least one of ChI or ChII, or extrachromosomal DNA.

Alterations

An “altered” chromosome is one that contains one or more of an insertion, deletion, substitution, rearrangement, inversion, or other genetic manipulation through human efforts and relative to the natural chromosome as found in Nature and unmodified by human activity. A substitution can also include the optimization of an existing gene. A deleted genetic element can also be relocated from ChII to ChI, or vice versa, or to extrachromosomal DNA. Thus, ChI can also be altered.

These alterations can be performed in any appropriate manner, e.g., by using insertion cassettes, or enzymatically through the use of a recombinase such as, for example, Cre-loxP system or Cre recombinase. The Cre recombinase can utilize known lox sites compatible with Cre recombinase (e.g., lox66 and lox71 sites). In other embodiments the alteration can be performed through the action of a nuclease such as, for example, Type II CRISPR Cas9. The alterations can also be performed with a homologous recombination vector or insertion cassette containing regions of sequence homology to a region in the genome where an insertion or deletion is desired. The homologous recombination vector can be incorporated by a single cross-over event or a double cross-over event. The alteration can also be performed through use of an integrase, such as, for example, PhiC31 or bxb1, or through the use of a suicide vector. In some examples the vector is assembled in vitro and subsequently transformed and amplified in E. coli. In some examples the vector is assembled in S. cerevisiae. In some examples, the amplified vector is introduced into the Vibrio sp. organism by conjugation, electroporation, chemical competence, biolistics, transduction, or via natural competence. Transformation of cells with any construct described herein can be performed by any suitable method, for example bacterial conjugation, electroporation, or chemical transformation.

In various embodiments of the organisms of the invention, in either of ChI, ChII, or the genome as a whole can be minimized, meaning one or more (or all) non-essential genetic elements (e.g. genes or other nucleic acid sequences) have been deleted, moved to the other chromosome or to extrachromosomal DNA, or otherwise removed to a substantial extent. For example a minimized chromosome can be reduced in size by at least 2.5% or at least 5.0% or at least 10% or at least 15% or at least 20% or at least 25% or at least 30% or at least 35% or at least 40% or at least 45% or at least 50%.

Genes are the basic unit of an inheritable trait in bacteria. Some genes can be further divided into regulatory elements, such as promoter/operators that regulate expression of the gene, and structural elements that comprise the coding portion. A genetic element refers to defined segments of DNA comprised in a cell's genome that perform a biological function within a cell. They can be naturally present or synthetic elements (added by human efforts). In some embodiments where Vibrio sp. is the host cell the genetic element is present on ChI or ChII and is not extrachromosomal DNA. Genetic elements can perform or support cellular functions, or encode proteins or peptides, or perform regulatory functions, bind ribosomes, or repair functions for nucleic acid sequences and can be part of the cellular machinery and/or replication machinery of the cell. Examples of genetic elements include, but are not limited to, a coding or non-coding gene, any sub-portion of a gene, a methyl-transferase encoding gene (e.g., Dam), a promoter, a termination sequence, a regulatory element, a 5′ or 3′ untranslated region, operators, a repeat, a control element, a protein or nucleic acid or ribosomal binding site, a transposon, or a structural “coding” portion of a gene, an initiation sequence (including, but not limited, to DnaA or RctB), multiple cloning sites, origins of transfer, conjugation or replication. In various embodiments a genetic element is involved in transcription, translation, recombination, the binding of a regulatory protein, the rate of transcription of a gene, or is a regulatory element. A “coding sequence” or “coding portion” refers to the portion of an mRNA or DNA molecule that codes for a polypeptide. It typically consists of the nucleotide residues of the molecule which are matched with an anticodon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon. The coding sequence may include nucleotide residues corresponding to amino acid residues which are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

An essential genetic element is one that, if not functionally present in the cell, results in the inability of the cell to replicate, survive, or maintain homeostasis in a specific environment in a substantially normal way. Some genetic elements are always essential, and some are only essential in a specific environment. Essential elements can be, but are not limited to, those required for a function such as basic metabolism needed for a cell to maintain homeostasis, DNA replication, transcription and/or translation of essential genes, proteins and peptides, functions necessary to maintain cellular structures, transport processes of essential materials into or out of the cell, or any combination thereof. Even essential genes or genetic elements can be deleted from ChII if moved to ChI or provided in trans.

Non-essential genetic elements can be identified experimentally, for example by using bioinformatics. Multiple wild types of Vibrio strain genomes can be evaluated and one can identify genes or nucleic acid sequences that are not consistently present in all strains. In other methods identification of non-essential genes can be achieved by transposon bombardment or other insertional mutagenesis screens that will produce multiple random integration mutants, and sequencing the genes disrupted in these viable mutants. Non-essential genes can also be sequentially removed by using homologous recombination. In some embodiments all essential genetic elements are comprised on Chromosome I or altered Chromosome II or extrachromosomal DNA. In another embodiment all essential elements are comprised on Chromosome I and no essential elements are comprised on Chromosome II or on extrachromosomal DNA. Non-limiting examples of non-essential genetic elements that can be deleted only from ChII or entirely from the organism include exonucleases, endonucleases, methylases, nucleases (e.g. Dns), restriction enzymes, partial or complete restriction-modification systems, mobile elements, phage proteins, a recombinase (e.g. recA), an endonuclease, a protease, a restriction enzyme, or any combination thereof. The non-essential genetic elements can be entirely deleted. In some embodiments all methylases can be deleted except for DAM methylases, which are required for chromosome replication. Additional genetic elements that can be deleted are also provides in FIG. 15. In a particular embodiment recA is deleted from ChI and/or the endonuclease Dns is deleted from ChI, where they are naturally present. In some embodiments all non-essential elements have been removed or deleted from ChI and ChII, but in other embodiments only most of the non-essential elements present on the natural chromosome have been removed or deleted from ChI and/or ChII.

Any of the organisms of the invention can comprise a deletion (or “knock out”) of certain genes or genetic elements. In some embodiments the organism has a deletion or knock out of a gene encoding endotoxin or of a regulatory element necessary for the transcription of endotoxin.

ChII-Based Vector

A problem encountered with cloning large pieces of DNA in an organism is that the total DNA load in the organism becomes too large and the organism cannot efficiently replicate or even survive with the amount of total cellular DNA. The present invention therefore deletes many or all non-essential or otherwise unnecessary genetic elements from a cell.

In some embodiments the engineered organisms of the invention have a ChI containing essential genetic elements, an altered ChII that is missing or has deleted at least one genetic element compared to natural ChII. In various embodiments the total size of the at least one genetic element(s) deleted or missing from the altered ChII are at least 10 kb or at least or at least 50 kb, or at least 100 kb, or at least 200 kb, or at least 300 kb, or at least 500 kb, or at least 700 kb, or at least 1 Mb or 10-50 kb or 10-100 kb or 10-200 kb or 10-300 kb or 50-100 kb or 50-200 kb or 50-300 kb or 50-500 kb or 50 kb-1 Mb or more than 1 Mb or more than 1.5 Mb or more than 2 Mb or the entire ChII. Missing or deleted elements can be moved to ChI or to extrachromosomal DNA. The then available “free” space ChII can be leveraged as a nucleic acid construct as a cloning vector, expression vector, shuttle vector, plasmid, cosmid, or artificial chromosome

The ChI and altered ChII together can have all essential elements of Vibrio sp., and the engineered organism can have all cellular and replication machinery necessary and functional for the cloning, replication and amplification of the heterologous DNA and altered ChII. During cell division or cellular growth, the exogenous DNA and altered ChII is therefore replicated and amplified. Cellular machinery refers to the physical and chemical components of a cell that function together to perform the physiological functions of the cell. In various embodiments the altered ChII can have less than 1.8 Mb or less than 1.6 Mb or less than 1.5 Mb or less than 1.3 Mb or less than 1.2 Mb or less than 1.0 Mb or less than 0.8 Mb of sequences present in the natural, unmodified ChII. The altered ChII can also have more than 2% or more than 3% or more than 5% or more than 10% or more than 25% or more than 50% or more than 75% of the sequences naturally present moved or deleted from ChB. These natural sequences that remain can be those that are naturally present in the organism and that function normally in the unmodified organism to conduct the physiological and other activities of the cell.

Thus, with nonessential elements deleted from ChII it can serve as a vector or plasmid or artificial chromosome carrying exogenous or heterologous DNA, for cloning, expression, or other purposes described herein. The heterologous or exogenous DNA comprised in ChII (or ChI) can comprise the sequence of a gene or other sequences, and can have a size of at least 10 kb, or at least 50 kb, or at least 100 kb, or at least 200 kb, or at least 300 kb, or at least 400 kb, or at least 500 kb, or at least 600 kb, or at least 700 kb, or at least 800 kb, or at least 900 kb, or at least 1 Mb, or at least 2 Mb, or 10 kb-200 kb, or 10 kb-300 kb, or 10 kb-500 kb, or 500 kb-1 Mb, or 300 kb-800 kb, or 10 kb-2 Mb, or 10 kb-1 Mb, or 10 kb-2 Mb, or 50 kb-500 kb, or 50 kb-1 Mb, or 50 kb-2 Mb, or 100 kb-1 Mb, or 100 kb-2 Mb, or 500 kb-1 Mb, or 500 kb-2 Mb, or 10 kb-3 Mb, or 50 kb-3 Mb, or 100 kb-3 Mb, or 500 kb-3 Mb. In some embodiments the heterologous or exogenous DNA will be a fragment of DNA that is a portion of a chromosome, or that expresses a protein of interest or other fragment of DNA that is to be cloned, amplified, or propagated.

In some embodiments an altered ChII can serve as a vector or plasmid or artificial chromosome having the exogenous or heterologous DNA and for the construction, cloning, maintenance, and/or recovery of large DNAs and for the expression, production, and secretion of proteins or peptides. The altered ChII can have genetic elements necessary for these function, including, for example, promoters, regulatory sequences, and/or signal sequences. In some embodiments the organism does not contain any extrachromosomal DNA. In some embodiments the organism does not contain any other exogenous or heterologous DNA other than that comprised on the altered ChII. But in other embodiments it can contain extra-chromosomal DNA as a natural plasmid and/or an optional exogenous plasmid, cosmid, artificial chromosome or an optional other vector. A natural plasmid or vector is one found in Vibrio sp. in any of the organism's natural environments and not present due to human efforts to place it there.

In some embodiments a plasmid, vector, or artificial chromosome of the invention comprising the DNA insert or essential genetic elements can be replicated and maintained in the host organism, which can be a Vibrio sp., an E. coli, a Bacillus subtilis, or a yeast.

“Exogenous nucleic acid molecule” or “exogenous gene” refers to a nucleic acid molecule or gene that has been introduced (“transformed”) into a cell. A transformed cell may be referred to as a recombinant cell or an engineered cell. A nucleic acid molecule is also exogenous if it is present in a descendent cell and received from an ultimate parent cell where that nucleic acid molecule was exogenous nucleic acid. The exogenous gene may be from a different species (thus also “heterologous”), or from the same species (thus “homologous”), relative to the cell being transformed.

The term “heterologous” when used in reference to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme refers to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is from a source or derived from a source other than the host organism species. Heterologous molecules are therefore always also exogenous, but exogenous molecules are not necessarily always heterologous. In contrast a “homologous” polynucleotide, gene, nucleic acid, polypeptide, or enzyme is used herein to denote a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is derived from the host organism species. When referring to a gene regulatory sequence or to an auxiliary nucleic acid sequence used for maintaining or manipulating a gene sequence (e.g. a promoter, a 5′ untranslated region, 3′ untranslated region, poly A addition sequence, intron sequence, splice site, ribosome binding site, internal ribosome entry sequence, genome homology region, recombination site, etc.), “heterologous” means that the regulatory sequence or auxiliary sequence is not naturally associated with the gene with which the regulatory or auxiliary nucleic acid sequence is juxtaposed in a construct, genome, chromosome, or episome. Thus, a promoter operably linked to a gene to which it is not operably linked to in its natural state (i.e. in the genome of a non-genetically engineered organism) is referred to herein as a “heterologous promoter,” even though the promoter may be derived from the same species (or, in some cases, the same organism) as the gene to which it is linked.

The altered Chromosome II can also comprise sequences of the natural Chromosome II. For example, it can contain a sequence of the replication machinery of the natural Chromosome II of Vibrio sp. The term “chromosomal replication machinery” or “replication machinery” refer to that part of an organism's chromosome which supports replication within the organism or in a different organism, and the replication machinery components can function together to perform the replication functions of the cell. In some embodiments replication machinery refers a 5.5 kb sequence from chromosome II of V. natriegens which is capable of supporting replication in an organism. In certain aspects, the replication machinery from V. natriegens can support replication in V. natriegens and E. coli. In some embodiments the sequence of replication machinery can have SEQ ID NO: 1 or a functional portion of SEQ ID NO: 1 or a variant of either. In some embodiments the SEQ ID NO: 1 can be operably linked to an exogenous nucleic acid sequence, which can also be heterologous. As used herein a “variant” of a nucleic acid or polypeptide sequence means having a sequence identity to a reference sequence (e.g. any of SEQ ID NO: 1-28) of at least 70% or at least 80% or at least 90% (and optionally less than 100%) or at least 95% (and optionally less than 100%) or at least 97% (and optionally less than 100%) or at least 98% (and optionally less than 100%) or at least 99% (and optionally less than 100%) or 90-99% or 90-95% or 95-99% or 97-99%. In other embodiments the variant has a sequence identity of at least 70% or at least 80% or at least 90% or at least 95% or at least 97% or at least 98% or at least 99% or 90-99% or 95-99% or 97-99% to a sequence of at least 10 or at least 15 or at least 20 or at least 30 or at least 40 or at least 50 or at least 100 or at least 200 or at least 300 or at least 400 or at least 500 or at least 600 or at least 700 or at least 800 or at least 900 or at least 1000 consecutive nucleotides or amino acids from the reference sequence (e.g. SEQ ID NO: 1-28). The invention also provides a genetically engineered nucleic acid molecule comprising any of SEQ ID NO: 1-28 or a variant of any of them. The nucleic acid molecule can also have an exogenous or heterologous sequence(s) on the 5′ and/or 3′ end, which exogenous or heterologous 5′ and 3′ sequence(s) can be compatible for cloning the nucleic acid molecule into a target vector (e.g., by homologous recombination).

As used herein, the terms “percent identity” or “homology” with respect to nucleic acid or polypeptide sequences are defined as the percentage of nucleotide or amino acid residues in the candidate sequence that are identical with the known polypeptides, after aligning the sequences for maximum percent identity and introducing gaps, if necessary, to achieve the maximum percent homology. N-terminal or C-terminal insertion or deletions shall not be construed as affecting homology, and internal deletions and/or insertions into the polypeptide sequence of less than about 30, less than about 20, or less than about 10 amino acid residues shall not be construed as affecting homology. Homology or identity at the nucleotide or amino acid sequence level can be determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx (Altschul (1997), Nucleic Acids Res. 25, 3389-3402, and Karlin (1990), Proc. Natl. Acad. Sci. USA 87, 2264-2268), which are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments, with and without gaps, between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified, and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases, see Altschul (1994), Nature Genetics 6, 119-129. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix, and filter (low complexity) can be at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff (1992), Proc. Natl. Acad. Sci. USA 89, 10915-10919), recommended for query sequences over 85 in length (nucleotide bases or amino acids).

For blastn, designed for comparing nucleotide sequences, the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N can be +5 and −4, respectively. Four blastn parameters can be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=1 (generates word hits at every winkth position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings for comparison of amino acid sequences can be: Q=9; R=2; wink=1; and gapw=32. A BESTFIT® comparison between sequences, available in the GCG package version 10.0, can use DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty), and the equivalent settings in protein comparisons can be GAP=8 and LEN=2. When referring to the polypeptide or nucleic acid sequences of the present disclosure, included are sequences considered to be derived from the original sequence, which have sequence identities of at least 40%, at least 45%, at least 50%, at least 55%, of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%, for example at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% or 90-99% or 95-99% or 97-99% or 98-99% sequence identity with the full-length polypeptide or nucleic acid sequence, or to fragments thereof comprising a consecutive sequence of at least 100, at least 125, at least 150 or more amino acid residues of the entire protein, or at least 100 or at least 200 or at least 300 or at least 400 or at least 500 or at least 600 or at least 700 or at least 800 or at least 900 or at least 1000 consecutive nucleotides; variants of such sequences, e.g., wherein at least one amino acid residue has been inserted N- and/or C-terminal to, and/or within, the disclosed sequence(s) which contain(s) the insertion and substitution. Contemplated variants can additionally or alternately include those containing predetermined mutations by, e.g., homologous recombination or site-directed or PCR mutagenesis, and the corresponding polypeptides or nucleic acids of other species, including, but not limited to, those described herein, the alleles or other naturally occurring variants of the family of polypeptides or nucleic acids which contain an insertion and substitution; and/or derivatives wherein the polypeptide has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid which contains the insertion and substitution (for example, a detectable moiety such as an enzyme).

As used herein, the phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms. (Schulz (1979) Principles of Protein Structure, Springer-Verlag). According to such analyses, groups of amino acids can be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure. (Schulz (1979) Principles of Protein Structure, Springer-Verlag). Examples of amino acid groups defined in this manner can include: a “charged/polar group” including Glu, Asp, Asn, Gln, Lys, Arg, and His; an “aromatic or cyclic group” including Pro, Phe, Tyr, and Trp; and an “aliphatic group” including Gly, Ala, Val, Leu, Ile, Met, Ser, Thr, and Cys. Within each group, subgroups can also be identified. For example, the group of charged/polar amino acids can be sub-divided into sub-groups including: the “positively-charged sub-group” comprising Lys, Arg and His; the “negatively-charged sub-group” comprising Glu and Asp; and the “polar sub-group” comprising Asn and Gln. In some examples, the aromatic or cyclic group can be sub-divided into sub-groups including: the “nitrogen ring sub-group” comprising Pro, His, and Trp; and the “phenyl sub-group” comprising Phe and Tyr. In another further example, the aliphatic group can be sub-divided into sub-groups including: the “large aliphatic non-polar sub-group” comprising Val, Leu, and Ile; the “aliphatic slightly-polar sub-group” comprising Met, Ser, Thr, and Cys; and the “small-residue sub-group” comprising Gly and Ala. Examples of conservative mutations include amino acid substitutions of amino acids within the sub-groups above, such as, but not limited to: Lys for Arg or vice versa, such that a positive charge can be maintained; Glu for Asp or vice versa, such that a negative charge can be maintained; Ser for Thr or vice versa, such that a free —OH can be maintained; and Gln for Asn or vice versa, such that a free —NH2 can be maintained. A “conservative variant” is a polypeptide that includes one or more amino acids that have been substituted to replace one or more amino acids of the reference polypeptide (for example, a polypeptide whose sequence is disclosed in a publication or sequence database, or whose sequence has been determined by nucleic acid sequencing) with an amino acid having common properties, e.g., belonging to the same amino acid group or sub-group as delineated above.

In various embodiments the altered ChII can also comprise an origin of replication operable in Vibrio sp. (e.g. R6Kγ or another) for the replication of DNA, and optionally a pir gene sequence or variant thereof encoding π (pi) protein or a variant thereof. The π (pi) protein or a variant thereof can also be provided in trans (e.g. on extrachromosomal DNA) when advantageous. The altered ChII can also comprise any one or more of suitable resistance genes such as an antibiotic (e.g. tetA/tetR, chloramphenicol) or another suitable marker, and/or one or more origin(s) of transfer (e.g., oriT from RP4, oriV, or other suitable origins of transfer), which can be used to facilitate mobilization of the vector via conjugation, or one or more multiple cloning sites with restriction enzyme cut sites; ARS/CEN sequences for replication in yeast, and a yeast selection marker (e.g. Trp gene or another suitable yeast selection marker). The vector can also, optionally, contain a copy control sequence. The vector can be transferred between Vibrio sp. and other species, for example between Vibrio sp. and E. coli or between Vibrio sp. and Bacillus subtilis or Vibrio sp. and yeast (e.g. Saccharomyces cerevisiae) or other species in either direction. Transfer can occur through bacterial conjugation or other suitable methods. The vector can also be transformed into Vibio sp., E. coli, Bacillus subtilis, or Saccharomyces cerevisiae cells by electroporation or by chemical transformation methods. Among other uses, the vector is useful in methods for cloning large DNA molecules in any of the aforementioned cells.

In addition to an origin of replication and a selection marker, ChI or ChII can also optionally have any one or more of an origin of transfer, a counter selection marker, a reporter gene, a regulatory element, an enzyme gene, or any combination of these. An origin of replication can be utilized from a variety of sources. In some embodiments the origin of replication is from the plasmid R6K. The gamma origin of replication from R6K (e.g., R6Kγ) can be utilized, but other origins of replication can also be utilized from other sources. When R6Kγ is the origin of replication it can be utilized with or without the pir gene encoding the pi (π) protein, which is necessary for plasmid replication. In other embodiments some or all of these elements can instead be provided on extrachromosomal DNA (e.g., a plasmid).

Selection markers can be utilized on the altered Chromosome II or on extrachromosomal DNA or any constructs described herein. In some embodiments the selection marker is a resistance gene, for example a gene conferring resistance to tetracycline, chloramphenicol, ampicillin, bleomycin, carbenicillin, gentamycin, glyphosate, hygromycin, kanamycin, neomycin, nourseothricin, phleomycin, puromycin, spectinomycin, streptomycin, or another antibiotic agent. In one embodiment the resistance gene is tetA/tetR. The resistance gene can also have an origin of replication from various sources, but in one embodiment is the RP4 oriT (which can be found on plasmid pJB3Tc20). The selection marker can also be ccdB.

In embodiments having a reporter gene, it can be a fluorescent protein or beta-galactosidase. In embodiments having an enzyme gene, it can be a recombinase, integrase, nuclease, recombineering enzymes, or polymerase. The recombinase can be Cre recombinase, the integrase can be PhiC31 or bxb1. The nuclease can be a Type II CRISPR Cas9 nuclease, and the polymerase can be a Sp6, T3, or T7 RNA polymerase. In some aspects, the vector is compatible with E. coli, V. natriegens, and/or S. cerevisiae.

Extra-chromosomal DNA (e.g. a plasmid) can be transformed into a cell by any suitable method, for example by bacterial conjugation (e.g. E. coli to Vibrio sp.), electroporation of electro-competent cells, chemical transformation into chemically competent cells, biolistics, transduction, or via natural competence. Efficiencies of transformation can be, for example, at least 1×10⁵ or at least at least 1×10⁶ at least 1×10⁷ at least 1×10⁸ cfu/ug DNA using any of the methods above.

Inducible Constructs

In some embodiments the Vibrio sp. organism of the invention has an inducible construct (e.g., a gene) engineered into ChI or ChII. The organism can also have a plasmid or other vector that has a nucleic acid sequence encoding an exogenous protein under the control of a promoter that is induced by the product of the inducible gene at ChI or ChII. Thus, when the inducer is provided to the cell transcription is initiated at the inducible gene, which produces a product that further induces transcription of the gene on the plasmid. In these embodiments the inducible gene can be any described herein and the promoter on the ChI or ChII can be any inducible promoter described herein. Also, the promoter on the plasmid or other vector can be any induced by the product of the inducible gene on ChI or ChII.

Inducible promoters used on any constructs described herein can include, but are not limited to, lacUV2 or lacUV5 promoter (which can be arabinose-inducible), a trc promoter (which is optionally regulated by lad and can be arabinose inducible), the araBAD promoter (which can be regulated by araC and arabinose inducible), the phase lambda pR promoter (which can be regulated by cI857 and be temperature inducible).

In some embodiments the construct is a plasmid and contains a gene of interest under the control of either an arabinose-inducible, IPTG-inducible, or temperature-inducible promoter (araBAD, trc, and pR promoter (controlled by cI857ts), respectively) and can be replicated either by the pBR322 or pl5a replication origins.

In some aspects, the genetically engineered Vibrio sp. of the invention further comprises a nucleic acid cassette having a heterologous nucleic acid sequence operably linked to a promoter, which can be a natural or a heterologous promoter. The heterologous promoter can be an inducible promoter, inducible by temperature, an aldopentose (e.g. arabinose) or IPTG, as non-limiting examples. In some examples the heterologous nucleic acid encodes T7 RNA polymerase.

In some aspects, the present invention provides a vector comprising Vibrio sp. chromosomal replication machinery. The replication machinery can contain any of the sequences described herein, e.g. SEQ ID NO: 1 or a variant thereof. In some examples, the vector also comprises a heterologous nucleic acid of interest. The vector can also have an inducible promoter operably linked to the nucleic acid of interest. In some embodiments the vector can be replicated in Vibrio sp., E. coli or S. cerevisiae. The invention also provides host cells, which can be any cell described herein. The host cells can comprise any nucleic acid or vector or expression cassette as described herein.

As used herein, “operably linked” is intended to mean a functional linkage between two or more sequences such that activity at or on one sequence affects activity at or on the other sequence(s). For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional link that allows for expression of the polynucleotide of interest. The term therefore refers to the positioning of a regulatory region and a coding sequence to be transcribed so that the regulatory region is effective for regulating transcription or translation of the coding sequence of interest. For example, to operably link a coding sequence and a regulatory region, the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the regulatory region. A regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by “operably linked” is intended that the coding regions are in the same reading frame. When used to refer to the effect of an enhancer, “operably linked” indicated that the enhancer increases the expression of a particular polypeptide or polynucleotides of interest. “Juxtaposed with” in the context of nucleic acid sequences, means the referenced sequences are part of the same continuous nucleic acid molecule.

Any of the organisms described herein can have a Chromosome I that contains a sequence encoding an inducible gene (e.g., T7 RNA polymerase or another inducible gene) although a non-inducible T7 RNA polymerase can also be used. The organism can also have an extra-chromosomal DNA that also has an inducible gene thereon that is transcribed when induced by the product of the inducible gene on ChI. Thus in one embodiment the extra-chromosomal DNA can have a T7 promoter or other suitable promoter recognized by T7 RNA polymerase (e.g. T3 or SP6) in front of a gene of interest. The extra-chromosomal DNA can be an RK2 plasmid containing an RK2 replicon, or can be any described herein. Using such a design the gene of interest can be transcribed (and translated) upon the production of T7 RNA polymerase or other inducible gene product from ChI. The inducible gene on ChI can be induced by any suitable molecule, as described herein. Therefore, under this design an extra-chromosomal DNA can be provided to a Vibrio cell that has an inducible gene on ChI. Upon being stimulated with the inducing molecule transcription occurs at the inducible gene. The product of the inducible gene triggers the promoter on the extra-chromosomal DNA and the exogenous DNA thereon is thus transcribed, producing the product of the gene of interest.

Signal Peptides

The engineered or recombinant Vibrio sp. of the invention can comprise an altered ChII or a plasmid, cosmid, artificial chromosome, or other vector that contains an exogenous or heterologous nucleic acid sequence that encodes an exogenous or heterologous protein or peptide, and a nucleic acid sequence encoding one or more functional Vibrio sp. signal peptides. The sequence encoding the functional signal peptide can be operably linked to the sequence encoding the protein or peptide. The signal peptide causes secretion of the expressed protein or peptide from the organism. The signal peptide can be expressed attached to the N-terminus or the C-terminus of the protein or peptide. The signal peptide can also be an internal signal sequence. The sequence encoding the protein or peptide can also be operably linked to a promoter (which can be exogenous, heterologous, or native) and/or other regulatory sequences. The protein or peptide can be secreted in various ways, for example through the Type III secretion system of Vibrio sp. In various embodiments the protein or peptide can be expressed from the altered ChII, from ChI, or from an exogenous vector, plasmid, cosmid, or artificial chromosome. In various embodiments the functional signal peptide can have a sequence of any of SEQ ID NO: 8-28 or a variant of any of them. A functional Vibrio sp. signal peptide is a sequence that causes the exogenous or heterologous protein or peptide to be secreted from a Vibrio sp. cell, which can be secreted into the periplasmic space or to the exterior of the organism. Signal peptides may be recognized by cellular structures or by the cell's secretory pathway that exports the protein or peptide from the cell. In some embodiments the sequences can be from 10-15 or 10-20 or 10-40 amino acids in length, or from 10-35 or from 10-25 or from 15-30 or from 15-30 or from 10-60 amino acids in length. In various embodiments the signal peptide can be any one or more of SEQ ID NO: 8-28 or a variant of any of them, or a functional fragment of any of them.

In various embodiments the organisms of the invention can secrete exogenous or heterologous proteins of interest in large amounts. The amount of exogenous or heterologous protein secreted can be expressed as mg of protein per liter of culture per time unit. The engineered organisms of the invention can secrete an exogenous or heterologous protein of interest in amounts of at least 1 mg/L/hour, or at least 5 mg/L/hour, or at least 10 mg/L/hour, or at least 20 mg/L/hour, or at least 50 mg/L/hour, or at least 70 mg/L/hour, or at least 100 mg/L/hour, or at least 200 mg/L/hour, or at least 300 mg/L/hour, or at least 500 mg/L/hour, or at least 1 g/L/hour, or 10-100 mg/L/hour, or 10-200 mg/L/hour, or 10-300 mg/L/hour, or 10-500 mg/L/hour, or 10 mg/L/hr to 1 g/L/hr or 10 mg/L/hr to 2 g/L/hr, or 50-100 mg/L/hour, or 50-200 mg/L/hour, or 50-500 mg/L/hour, or 50 mg/L/hr to 1 g/L/hr or 50 mg/L/hr to 2 g/L/hr.

The proteins or peptides that are expressed, produced, and secreted in the invention can be any protein or peptide, for example insulin, pro-insulin, glucagon-like peptide, a gonadotropin releasing hormone, an agonist of gonadotropin releasing hormone, somatostatin and inhibitors thereof, octreotide, goserelin, leuprolide, granulocyte stimulating factor (rh-GCSF), levansucrase, as some examples.

Shuttle Vector

The altered ChII of the invention can contain genetic elements that enable it to be replicated or used as a cloning or expression vector not only in Vibrio sp., but also in E. coli. or Bacillus sp., (e.g., Bacillus subtilis), or in E. coli and Bacillus sp. The altered ChII can therefore serve as a shuttle vector between Vibrio sp. and E. coli, or between Vibrio sp. and Bacillus sp., or between E. coli and Bacillus sp. In various embodiments the shuttle vector comprises a sequence from ChII, an origin of replication for each cell type (a Vibrio organism and a non-Vibrio organism, e.g., E. coli), and one or more selection markers. It can have any of the promoters described herein, for example lambda phage pR, pL, trc, lacUV5, araBAD, or a tet-inducible promoter.

The shuttle vector of the present invention contains the replication machinery from Vibrio sp. In one embodiment the replication machinery of Vibrio sp. has SEQ ID NO: 1 or a variant thereof. Shuttle vectors can carry any DNA of interest. Examples include DNA encoding for a protease, a phytase, a metabolic enzyme, proinsulin, or a granulocyte colony stimulating factor (GCSF). The DNA can also encode for an entire enzymatic pathway or a partial or complete bacterial chromosome.

Cold Tolerance

Reactive oxygen species (ROS) such as singlet oxygen, superoxide anion, hydrogen peroxide, and hydroxyl radicals are a consequence of aerobic metabolism and can cause cellular damage through the oxidation of biological molecules. These oxygen species can be generated in an enhanced amount as a result of various types of cellular stress, including cold stress. The invention provides an engineered Vibrio sp. organism comprising one or more nucleotide sequence(s) encoding one or more enzyme(s) from an ROS detoxification system. The one or more enzyme can be selected from one or more of a peroxidase, a dismutase, a reductase, and a transferase, or any combination of them, and which enzyme can be an algal, microalgal, bacterial, cyanobacterial or other type or source of enzyme. The enzyme can be selected from one or more of glutathione peroxidase (which can have reduced monomeric glutathione (GSH) as substrate), superoxide dismutase, guaiacol peroxidase (GPX), enzymes of ascorbate-glutathione (AsA-GSH) cycle ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), catalase peroxidase (e.g. katG and/or katE), alkyl hydroperoxide reductase, and glutathione S-transferase. The nucleotide sequence(s) can be exogenous or heterologous, and the one or more enzymes can be exogenous or heterologous enzymes.

The invention provides engineered Vibrio sp. organisms having a heterologous or exogenous nucleic acid sequence encoding at least one enzyme from an ROS detoxification system, which can be present on a plasmid or other vector. It was discovered unexpectedly that the engineered organisms have a greater ability to tolerate cold stress than non-engineered organisms and can therefore remain viable and culturable after incubation at lower temperatures and for substantially greater periods of time than non-engineered organisms. An ROS detoxification system can convert any of the reactive oxygen species into one or more of oxygen or water. The enzyme from an ROS system can be operably linked to or under the control of an exogenous or heterologous promoter and/or other regulatory sequences.

Some organisms are cold sensitive, meaning that when stored or incubated at lower temperatures they go into a non-viable or non-culturable state when incubated at low temperatures (e.g., below 6° C. or below 5° C. or below 4° C.) for as little as 2-3 days and cannot be grown up or amplified efficiently to produce new colonies. Natural Vibrio sp. are cold sensitive organisms. In one embodiment the engineered Vibrio sp. organism is maintained on a solid media, i.e. one that does not flow when tilted against gravity. But liquid or semi-solid media can also be used depending on the application. Some solid media contain 0.6% or more, or at least 0.7% or at least 0.8% or at least 0.9% or at least 1.0% or at least 1.1% or at least 1.2% or at least 1.3% or at least 1.4% agar. Semi-solid media are soft enough so that motile bacteria can swim through it. An example of semi-solid media would be one having less than 0.6% agar.

In some embodiments the Vibrio sp. of the invention is able to remain viable and culturable after incubation or storage at temperatures of less than 20° C. or less than 15° C. or less than 10° C. or less than 8° C. or less than 6° C. or less than 5° C., or less than 4° C., or less than 3° C., or less than 2° C., or less than 1° C. or 0-4° C. or 0-5° C. or 0-6° C. or 1-4° C. or 1-5° C. or 1-6° C. or 3-5° C. or 2-6° C. The organism can be stored or incubated at these temperatures for a least 1 hour or at least 2 hours or at least 6 hours or at least 12 hours or at least 24 hours or at least 48 hours or at least 3 days or at least 5 days or at least 7 days or at least 9 days or at least 10 days or at least 12 days or at least 14 days or at least 16 days or at least 18 days or at least 19 days or at least 20 days or at least 22 days or at least 24 days or at least 26 days or at least 28 days or at least 30 days and remain culturable and/or viable. In some embodiments the organism retains viability or culturability after storage or incubation at the stated temperatures and the stated time periods on solid medium as described above or in a liquid broth or semi-solid medium. Viability or culturability refers to the ability of a colony to generate new colonies after incubation at low temperatures described herein and re-streaking. This can be determined by touching a colony and spreading it on a culture plate and determining the number of colonies that are generated. Non-viable or non-culturable colonies produce substantially fewer or no colonies while culturable or viable colonies produce a normal number of colonies and/or can show robust growth after such incubations. In other embodiments the engineered organism remains substantially more viable or more culturable than non-engineered organisms after being cultivated at low temperature under the same conditions. The engineered organisms can produce at least 2× or at least 3× or at least 5× or at least 10× or at least 20× or at least 30× or at least 40× or at least 50× or at least 100× as many colonies as non-engineered organisms when re-cultured under the same conditions.

In various embodiments the cold tolerant Vibrio sp. of the invention is a Vibrio sp. described herein. Cold tolerance can be conferred by engineering one or more exogenous or heterologous nucleic acid sequences conferring cold tolerance, as described herein, into a vector (e.g. a plasmid, cosmid, artificial chromosome or other vector), or into ChI or ChII or a combination thereof. The organism can be transformed with the vector. In some embodiments the engineered Vibrio sp. organism contains an exogenous or heterologous nucleotide sequence encoding at least one alkyl hydroperoxide reductase gene (e.g. an ahp operon), or a catalase gene katG and/or katE, or a glutathione S-transferase gene, or any combination of these. The sequences can be under the control of or operably linked to a promoter and/or other appropriate regulatory sequences, which can be exogenous or heterologous. The nucleic acid sequences can be operable in Vibrio sp. and in other organisms as well. In embodiments where the alkyl hydroperoxide reductase operon is present it can be the ahpCF from E. coli or a variant thereof. In embodiments where the catalase peroxidase gene is present it can be katG or katE, but both can also be used. katG and katE can be obtained from E. coli or other sources. In embodiments where the glutathione S-transferase gene is present it can be gstA, and can be from E. coli or another suitable source. FIG. 10a-c provides non-limiting examples of vectors for expressing cold tolerance.

In one embodiment the vector or construct is a plasmid and has a p15A origin of replication, a tetracycline resistance cassette, the origin of transfer for plasmid RP4, and E. coli katE, katG, or the ahpCF operon, optionally under the control of an arabinose-inducible promoter (as described herein) or another promoter.

Expression Cassettes

In another aspect the invention provides expression cassettes operable in Vibrio sp. that has a promoter inducible with sucrose. The promoter can be operably linked to a heterologous gene that encodes a protein or peptide or other desirable nucleic acid molecule. The expression cassette can further encode a signal peptide that causes the secretion of the heterologous protein or peptide from the organism. The inducible promoter, and the sequences encoding the signal peptide and heterologous protein or peptide can all be operably linked so that upon inducing the promoter the organism produces and secretes the heterologous protein or peptide. The heterologous protein or peptide can be secreted with the signal peptide attached or the signal peptide can be removed by the organism and secrete the heterologous protein or peptide without the signal peptide. The signal peptide can be any described herein. In one embodiment the inducible promoter has SEQ ID NO: 5 or 6 or a portion or variant of SEQ ID NO: 5 or 6. When the inducible promoter comprises a portion of SEQ ID NO: 5 or 6 it can comprise at least 50% or at least 60% or at least 70% or at least 80% or at least 90% or at least 95% or at least 97% or at least 98% or at least 99% of SEQ ID NO: 5 or 6.

In some embodiments the inducible promoter has a sequence identity of at least 70% or at least 80% or at least 90% or at least 95% or at least 97% or at least 98% or at least 99% of any of SEQ ID NO: 5-6, or a variant thereof. In other embodiments the inducible promoter has a sequence identity of at least 70% or at least 80% or at least 90% or at least 95% or at least 97% or at least 98% or at least 99% to a sequence of at least 100 or at least 200 or at least 300 or at least 400 or at least 500 consecutive nucleotides from SEQ ID NO: 5 or 6.

The expression cassettes can be comprised in any vector operable in Vibrio sp. The expression cassette can also include a 3′ untranslated region (e.g., a poly-A sequence) and/or a 5′ untranslated region (or leader sequence).

“Expression cassette” as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to a nucleotide sequence of interest, which can optionally be operably linked to termination signals and/or other regulatory elements. An expression cassette can also have sequences that enable, mediate, or enhance translation of the nucleotide sequence. The coding region usually codes for a protein or peptide of interest but may also code for a functional RNA of interest, for example antisense RNA or a non-translated RNA, in the sense or antisense direction. An expression cassette may be assembled entirely extracellularly (e.g., by recombinant cloning techniques). However, an expression cassette may also be assembled using in part endogenous components. For example, an expression cassette may be obtained by placing (or inserting) a promoter sequence upstream of an endogenous sequence, which thereby becomes functionally linked and controlled by said promoter sequences. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus. An expression cassette can comprise one or more sequences, or even only sequences, that are heterologous to the host cell in which it is expressed or carried. An “expression vector” is a vector that comprises an expression cassette. A “vector” is any genetic element capable of serving as a vehicle of genetic transfer, expression, or replication for a foreign polynucleotide in a host cell. For example, a vector may be an artificial chromosome or a plasmid, and may be capable of stable integration into a host cell genome, or it may exist as an independent genetic element (e.g., episome, plasmid). A vector may exist as a single polynucleotide or as two or more separate polynucleotides. Vectors may be single copy vectors or multicopy vectors when present in a host cell.

The term “promoter” refers to a nucleic acid sequence capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. A promoter includes the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. A promoter can include a transcription initiation site as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Prokaryotic promoters may contain −10 and −35 prokaryotic promoter consensus sequences. A promoter may be a constitutive promoter, a repressible promoter, or an inducible promoter. Some promoters are synthetic sequences and are not naturally-occurring sequences. Promoters can be endogenous or exogenous promoters.

Kits

In some aspects, the present invention provides a kit for cloning DNA. In one embodiment the kit can contain an engineered Vibrio sp. cell disclosed herein. In some embodiments the kit can contain a vector comprising Vibrio sp. chromosomal replication machinery, as disclosed herein. The kit can also have host cells disclosed herein that are transformable with the vector. The kit can also contain a buffer compatible with the host cells. Optionally, the kit can also contain information with instructions from cloning DNA or for producing a protein or peptide, or information directing the user to an internet page containing instructions for cloning DNA or for producing the protein or peptide. The kits of the invention can contain any one or more of the above components in any combination of the components. The host cells provided with the kit can optionally contain the vector of the kit. The host cells can also optionally be provided in a container having the buffer provided with the kit or on a solid or semi-solid media. The vector provided in the kit can optionally have an inducible promoter described herein. In various embodiments the host cells are any Vibrio sp. bacteria disclosed herein, but the host cells can also be E. coli or S. cerevisiae or Bacillus cells.

The following examples are provided to further illustrate the advantages and features of the present invention, but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

Example 1 Growth of V. natriegens in a Range of Growth Media as Well as at Multiple Temperatures

Growth of V. natriegens was examined on a number of different growth media and at multiple temperatures. A glycerol stock of V. natriegens was used to inoculate liquid cultures or was streaked out on agar plates. Liquid cultures were cultivated with agitation ranging from 175-220 RPM at the indicated temperatures. After overnight incubation, plates/cultures were examined for growth. Growth was defined as turbidity (in the case of liquid cultures) or visible colonies (in the case of agar plates).

Media compositions are as follows:

LB broth: 10.0 g/L Tryptone, 5.0 g/L Yeast Extract, 10.0 g/L NaCl

LB broth+v2 salts: LB broth supplemented with additional salts (204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2)

LB broth+v2 salts+glucose: LB broth supplemented with additional salts (204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2)+0.2% glucose

LB broth+v3 salts: LB broth supplemented with additional salts (475 mM NaCl, 9.7 mM KCl, and 54 mM MgCl2)

LB broth+v3 salts+glucose: LB broth supplemented with additional salts (475 mM NaCl, 9.7 mM KCl, and 54 mM MgCl2)+0.2% glucose

LB agar: LB media+1.5% agar-agar

LB agar+v2 salts: LB broth supplemented with additional salts (204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2)+1.5% agar-agar

LB agar minus NaCl with 6% sucrose: 10.0 g/L Tryptone, 5.0 g/L Yeast Extract, 1.5% agar-agar, 6% sucrose

Nutrient Broth+1.5% NaCl: 8 g/L DIFCO™ Nutrient Broth (Cat. No. 234000) supplemented with 1.5% NaCl

Nutrient Agar+1.5% NaCl: 8 g/L DIFCO™ Nutrient Broth (Cat. No. 234000) supplemented with 1.5% NaCl and 1.5% agar-agar

Brain Heart Infusion Broth: 37 g/L Teknova Brain Heart Infusion Broth

Brain Heart Infusion Broth+2% NaCl: 37 g/L Teknova Brain Heart Infusion Broth Dry Media (Cat. No. B9500)+20 g/L NaCl

Brain Heart Infusion Broth+1.5% Instant Ocean: 37 g/L Teknova Brain Heart Infusion Broth Dry Media (Cat. No. B9500)+15 g/L Instant Ocean Sea Salt Mixture

Brain Heart Infusion Broth+v2 salts: 37 g/L Teknova Brain Heart Infusion Broth Dry Media (Cat. No. B9500) supplemented with additional salts (204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2)

Brain Heart Infusion Broth+v3 salts: 37 g/L TEKNOVA Brain Heart Infusion Broth Dry Media (Cat. No. B9500) supplemented with additional salts (475 mM NaCl, 9.7 mM KCl, and 54 mM MgCl2)

Brain Heart Infusion Agar+1.5% Instant Ocean: 52 g/L Difco™ Brain Heart Infusion Agar (Cat. No. 241830)+15 g/L Instant Ocean Sea Salt Mixture

Brain Heart Infusion Agar: 37 g/L Teknova Brain Heart Infusion Broth Dry Media (Cat. No. B9500)+1.5% agar-agar

Brain Heart Infusion Agar+v2 salts: 37 g/L Teknova Brain Heart Infusion Broth Dry Media (Cat. No. B9500)+1.5% agar-agar supplemented with additional salts (204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2)

M9 glucose media (500 mL): 1× M9 Salts, 0.4% glucose, 2 mM MgSO₄, 0.1 mM CaCl₂

M9 glucose agar: M9 glucose media supplemented with 1.5% agar-agar

M9 glucose with 1% sucrose: 1× M9 Salts, 0.4% glucose, 2 mM MgSO₄, 0.1 mM CaCl₂, 1% sucrose

M9 glucose with 2% sucrose: 1× M9 Salts, 0.4% glucose, 2 mM MgSO₄, 0.1 mM CaCl₂, 2% sucrose

M9 glucose with 4% sucrose: 1× M9 Salts, 0.4% glucose, 2 mM MgSO₄, 0.1 mM CaCl₂, 4% sucrose

M9 1% sucrose: 1× M9 Salts, 2 mM MgSO₄, 0.1 mM CaCl₂, 1% sucrose

M9 2% sucrose: 1× M9 Salts, 2 mM MgSO₄, 0.1 mM CaCl₂, 2% sucrose

M9 4% sucrose: 1× M9 Salts, 2 mM MgSO₄, 0.1 mM CaCl₂, 4% sucrose

marine agar: 55.1 g/L Difco™ Marine Agar 2216 (Cat. No. 212185)

Bacto Heart Infusion Broth: 25 g/L Bacto™ Heart Infusion Broth (Cat. No. 238400)

SSG agar: 28 g/L Bacto™ SOB Medium (Cat. No. 244310), 17% Fetal Bovine Serum, 1% glucose, 4 mL/L Phenol Red Solution (Sigma P0290)

2×YT+v2 salts+glucose+phosphate buffer: 2×YT media (16 g/L Tryptone, 10 g/L Yeast Extract, 5 g/L NaCl) is supplemented with v2 salts (204 mM NaCl, 4.2 mM KCl, 23.14 mM MgCl₂), 17.61 mM Na₂HPO₄, 0.2% glucose. pH is adjusted to 7.4.

Vegitone Infusion Broth+v2 salts: Vegitone Infusion Broth (Sigma Aldrich cat #41960) supplemented with v2 salts (204 mM NaCl, 4.2 mM KCl, 23.14 mM MgCl₂).

LB+v2 salts+glucose+phosphate buffer: LB media (10 g/L Tryptone, 5 g/L Yeast Extract, 10 g/L NaCl) is supplemented with v2 salts (204 mM NaCl, 4.2 mM KC1, 23.14 mM MgCl₂), 17.6 mM K₂HPO₄, 0.2% glucose. pH is adjusted to 7.0.

Results of the growth experiments are presented in Table 1:

TABLE 1 Media Format 25° C. 30° C. 37° C. LB broth liquid Growth growth no growth culture LB broth + v2 salts liquid N/A growth growth culture LB broth + v2 salts + glucose liquid N/A growth growth culture LB broth + v3 salts liquid N/A growth growth culture LB broth + v3 salts + glucose liquid N/A growth growth culture LB agar agar plate Faint growth growth LB agar + v2 salts agar plate N/A growth growth LB agar minus NaCl with agar plate N/A no no growth 6% sucrose growth Nutrient Broth + 1.5% NaCl liquid Growth growth no growth culture Nutrient Agar + 1.5% NaCl agar plate slow growth attenuated growth growth Brain Heart Infusion Broth liquid Growth growth attenuated culture growth Brain Heart Infusion Broth + liquid Growth growth growth 2% NaCl culture Brain Heart Infusion Broth + liquid Growth growth growth 1.5% Instant Ocean culture Brain Heart Infusion Broth + liquid N/A growth growth v2 salts culture Brain Heart Infusion Broth + liquid N/A growth growth v3 salts culture Brain Heart Infusion Agar + agar plate slow growth growth 1.5% Instant Ocean growth Brain Heart Infusion agar agar plate N/A growth N/A Brain Heart Infusion agar + agar plate Growth growth growth v2 salts M9 glucose media liquid slow growth N/A culture growth M9 glucose agar agar plate slow growth slow growth growth M9 glucose with 1% sucrose agar plate N/A growth growth M9 glucose with 2% sucrose agar plate N/A growth growth M9 glucose with 4% sucrose agar plate N/A growth growth M9 1% sucrose agar plate N/A growth growth M9 2% sucrose agar plate N/A growth growth M9 4% sucrose agar plate N/A growth growth marine agar agar plate slow growth growth growth Bacto Heart Infusion Broth liquid N/A growth attenuated culture growth SSG agar agar plate Growth growth growth 2xYT + v2 salts + glucose + Liquid N/A growth growth phosphate buffer culture Vegitone Infusion Broth + Liquid N/A growth growth v2 salts culture LB + v2 salts + glucose + Liquid N/A growth growth phosphate buffer culture

Example 2 Transformation of V. natriegens with Exogenous DNA Constructs Via Conjugation

This method was used to transfer a mobilizable plasmid from E. coli into V. natriegens where:

1) the plasmid was maintained as an episomal molecule in V. natriegens, or

2) where (with appropriate plasmid design) the plasmid integrated into the V. natriegens chromosome via a single or double-crossover integration event.

Donor preparation: 10 mL of LB medium containing appropriate antibiotic was inoculated with E. coli donor strain (containing mobilizable plasmid of interest) and incubated overnight at 37° C. with agitation (200 RPM). Acceptable donor strains include, but are not limited to strain S17-1 λpir (containing the RP4 conjugation machinery integrated into the chromosome) or EPI300 cells harboring the pRL443 conjugative plasmid.

Recipient preparation: 10 mL of LB medium was inoculated with V. natriegens recipient strain and incubated overnight at room temperature with agitation (175 RPM).

Conjugative mating: Donor and recipient cultures were retrieved from incubators. 1 mL of each culture was separately centrifuged at 5000×g for 3 min in a 1.5 mL Eppendorf tube to pellet the cells. The supernatant was decanted and the cell pellets were each resuspended in 1 mL fresh LB medium. The wash (centrifugation/decanting/resuspension) was repeated for the donor strain to further reduce residual antibiotic carryover. Donor and recipient cultures were then mixed in multiple different ratios (e.g., 1:9, 1:4, 1:1, 4:1, 9:1 donor:recipient) in a total volume of 100 μL. The 100 μL of cells were spotted out as 10 μL spots on prewarmed LB plates, and incubated at 30° C. for 3-5 hours. Cells were washed from plate with 1 mL M9 glucose medium. Various volumes of cells (1 μL, 5 μL, 20 μL) were plated out on M9 glucose plates containing appropriate antibiotic and incubated overnight at 30° C. The E. coli donor strains mentioned above will not grow on the M9 medium utilized for this procedure (see recipe below). Individual V. natriegens colonies that grew on the M9 selective plate were then screened for successful conjugation event via standard methods.

M9 glucose medium (500 mL):

100 mL 5× M9 Salts

390 mL ddH2O

7.5 g agar-agar*

10 mL 20% glucose**

1 mL 1 M MgSO₄**

50 μL 1 M CaCl₂**

*for solid media, add agar-agar

**added after autoclaving

Example 3 Transformation of V. natriegens with Exogenous DNA Constructs Via Electroporation

Preparation of Electrocompetent cells: 10 mL of Brain Heart Infusion Broth supplemented with supplemented salts (204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2) was inoculated with Vibrio natriegens and incubated overnight at 30° C. with agitation. On the following day, 250-500 mL of the same growth media was inoculated with the overnight culture at a dilution of 1:100 to 1:200 (overnight culture:fresh media). The culture was grown at 37° C. with shaking until an OD600 of 0.5. The culture was then split into two pre-chilled 250 mL centrifuge bottles which were then incubated on ice for 0-20 mM The cells were pelleted at 6500 RPM in a JA-14 centrifuge rotor for 20 mM at 4° C. The supernatant was carefully decanted and the cell pellets were gently resuspended in 5-10 mL of electroporation buffer (680 mM sucrose, 7 mM K₂HPO₄, pH 7). The suspension was transferred to centrifuge tubes and the tube was filled to top (˜35 mL) with additional electroporation buffer and inverted several times to mix. The cells were spun down at 6750 RPM for 15 mM at 4° C. in a JA-17 rotor. The supernatant was decanted with pipette. The wash was repeated two times for a total of three washes. After the final wash, the cells were gently resuspended in residual electroporation buffer. The volume was adjusted with additional electroporation buffer to bring the final OD600 to 16. Cells were aliquoted into pre-chilled tubes and were stored at −80° C. until use.

Electroporation protocol: A vial of competent cells was retrieved from storage at −80° C. and allowed to thaw on ice. Plasmid DNA and electrocompetent cells were combined and gently mixed in a pre-chilled 1.5 mL microfuge tube. The cell/DNA suspension was transferred to a pre-chilled electroporation cuvette with a 0.1 cm gap size. Cells were electroporated with the following parameters: 700-900 V, 25 μF, 200 S2, 1 mm cuvette. Cells were immediately recovered in 500 μL recovery media (Brain Heart Infusion Broth supplemented with supplemented salts (204 mM NaCl, 4.2 mM KCl, 23.14 mM MgCl2, and 0-680 mM sucrose) and transferred to a 15 mL culture tube. The cells were recovered by incubating at 30-37° C. for 1-2 hours. Aliquots of the recovery media were plated out on pre-warmed agar plates containing appropriate antibiotic. Acceptable agar media include, but are not limited to: M9 glucose, Brain Heart Infusion Agar (with or without additional salt supplementation), and LB (with our without additional salt supplementation). The plates were incubated for several hours to overnight at 30-37° C. for colonies to appear.

Example 4 The Use of V. natriegens as a Host for Molecular Cloning

Recombinant DNA fragments for assembly were derived from multiple sources including, but not limited to: digestion of existing recombinant DNA using nucleases (e.g., restriction enzymes, homing endonucleases, zinc-finger nucleases, TALENs, Cas9 nuclease with appropriate guide RNAs, etc.), PCR amplification, or de novo gene assembly from synthesized oligonucleotides.

In vitro assembly was carried out with any number of standard DNA construction techniques or commercially available kits including, but not limited to: ligation of DNA fragments using a suitable DNA ligase and Gibson Assembly. Alternatively, in vivo assembly can be performed in a compatible host cell, such as, for example, E. coli or S. cerevisiae followed by isolated of the assembled product

Once in vitro or in vivo assembly and isolation, if appropriate, is complete, V. natriegens competent cells that have been prepared according to the conjugation or electroporation protocol were transformed according to the appropriate protocol in either Example 2 or Example 3. Cells were plated on agar plates containing the appropriate antibiotic and incubated for several hours to overnight at 30-37° C. for colonies to appear.

Colonies isolated from agar plates containing appropriate antibiotics were used to inoculate growth media containing the same antibiotic. Cells were grown for ˜3 hours to overnight at 30-37° C. Cells were harvested by centrifugation, and DNA was then extracted via standard methods (e.g., alkaline lysis techniques) or commercially available kits (e.g., QIAspin Miniprep Kit from Qiagen). Extracted DNA was analyzed by standard methods.

Example 5 The Use of V. natriegens as a Host for Inducible Protein Expression

A series of plasmids were designed for inducible protein expression of green fluorescent protein (GFP) in various experiments. The plasmids were designed to contain:

1) one of three promoters (the IPTG-inducible trc promoter, arabinose-inducible araBAD promoter, or the temperature inducible λ pR promoter modulated by the temperature-sensitive cI857 repressor.

2) one of two origins of replication (the p15a origin of replication or the origin from plasmid pBR325)

3) a green fluorescent protein (GFP) under the control of the inducible promoter to monitor expression

4) a transcriptional terminator following GFP (rrnB transcriptional termination sequence)

5) a chloramphenicol resistance gene for antibiotic selection

The functional elements and their source plasmids are listed in Table 2:

TABLE 2 element source plasmid trc promoter pTrcHisA araBAD promoter pKD46 λ pR promoter/temperature- 705-cre sensitive cI857 repressor p15a origin pACYC184 pBR325 origin pBR325 GFP gene synthesized from oligos rrnB transcriptional termination pTrcHisA sequence chloramphenical acyl transferase pCC1BAC gene

The maps for the six plasmids are shown in FIG. 1. The plasmids were assembled in vitro using Gibson Assembly and electroporated into V. natriegens following the protocols described in Example 4.

Cultures of individual transformed colonies were grown overnight in LB media (10.0 g/L Tryptone, 5.0 g/L Yeast Extract, 10.0 g/L NaCl) supplemented with additional salts (204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2) at 30° C. with agitation at 200 RPM. On the following day the cultures were used to inoculate fresh salt-supplemented LB media at a ratio of 1:100 overnight culture:fresh media. The cultures were grown at 30° C. with agitation until an OD600 of 0.5. Cultures were then induced with appropriate inducer (0.2% arabinose, 1 mM IPTG, or shifting temperature to 42° C., for the araBAD, trc, and pR promoters respectively). After about 4 hours, the OD600 and GFP fluorescence (excitation 480 nm/emission 510 nm) were measured. FIG. 2 shows GFP fluorescence normalized to OD600 for induced and non-induced cultures harboring each of the six expression plasmids. The data demonstrates the functionality of these inducible promoter systems in this species.

For further analysis, cultures of V. natriegens harboring pBR322-trc-GFP (induced and non-induced) were collected via centrifugation, resuspended in lysis buffer (20 mM Tris pH 8, 2 mM MgCl₂), lysed via sonication, clarified via centrifugation, and run on a 4-12% 10-well BOLT® Bis-Tris gel (LIFE TECHNOLOGIES®) with MES running buffer, which was subsequently stained with SIMPLYBLUE™ safe stain (LIFE) TECHNOLOGIES®. In FIG. 3, Lane 1: SEEBLUE™ Plus2 Protein Standard (LIFE) TECHNOLOGIES®; Lane 2: 1 μL V. natriegens pBR322-trc-GFP lysate from IPTG-induced culture; Lane 3: 10 μL V. natriegens pBR322-trc-GFP lysate from IPTG-induced culture; Lane 4: 1 μL V. natriegens pBR322-trc-GFP lysate from non-induced culture and Lane 5: 10 μL V. natriegens pBR322-trc-GFP lysate from non-induced culture. The GFP protein can be seen in the lanes corresponding to the induced culture, but is present at much lower levels in the non-induced culture.

Example 6 The Use of ChII as a Cloning/Shuttle Vector in Vibrio or Non-Vibrio Species (e.g., E. coli)

The replication machinery of V. natriegens ChII comprises SEQ ID NO: 1.

The vector designated pVnatoriCII was prepared by assembling the following DNA regions from the following sources:

1) V. natriegens chrII sequence (amplified from V. natriegens genomic DNA) (SEQ ID NO: 1);

2) R6Kγ origin of replication (amplified from plasmid pR6Kan from EPICENTRE®) (SEQ ID NO: 2); and

3) tetA/tetR resistance genes+RP4 oriT (amplified from plasmid pJB3Tc20) (SEQ ID NO: 3).

The PCR primers were designed to generate sufficient homology overlaps between the PCR products to facilitate vector construction via GIBSON ASSEMBLY® to generate the plasmid shown in FIG. 4.

The plasmid comprises the sequence from V. natriegens ChII, the R6Kγ origin of replication (without the pir gene encoding the pi π protein necessary for plasmid replication) and the tetA/tetR resistance genes (as a selective marker) along with the RP4 oriT region (to facilitate mobilization of plasmid via conjugation) of plasmid pJB3Tc20 (NCBI genbank U75324).

The vector was assembled in vitro according to standard methods and was transformed into EC100D pir-116 E. coli cells from EPICENTRE® via electroporation. These cells contain the pir gene encoding the pi π protein necessary for replication of plasmids containing the R6Kγ origin of replication. Because the designed plasmid contains the R6Kγ origin, the plasmid will be able to replicate in this strain regardless of the functionality of the V. natriegens chrII machinery in E. coli. Cells were plated out on LB agar plates containing 10 μg/mL tetracycline. Individual colonies were grown up in LB media containing 10 μg/mL tetracycline, and DNA was recovered using the QIAPREP® Spin Miniprep Kit from QIAGEN®. Proper vector assembly was verified via restriction digestion analysis. Plasmids with the correct restriction pattern were then electroporated into EPI300 E. coli cells from EPICENTRE® via electroporation. Because EPI300 cells do not contain the pir gene encoding the pi π protein necessary for replication of plasmids containing the R6Kγ origin of replication, the only way this plasmid will replicate is if the V. natriegens ChII replication machinery is able to support plasmid replication in E. coli. The transformation of EPI300 cells with pVnatoriCII resulted in tetracycline resistant colonies, indicating the plasmid successfully replicated in this strain.

Conjugation Mediated Transfer of pVnatoriCII to from E. coli to V. natriegens

The plasmid was also transferred to V. natriegens via conjugation from E. coli strain S17-1 λpir following the conjugation protocol described in Example 2, giving rise to tetracycline-resistant V. natriegens colonies.

Cloning Large DNAs into pVnatoriCII:

To assess the utility of pVnatoriCII for harboring large DNA molecules in E. coli, we cloned an about 135 kb sequence from the Mycoplasma genitalium genome into the plasmid (by replacing the R6Kγ origin with M. genitalium sequence) to generate plasmid pVnoriCII-Mgen25-49. A plasmid carrying a fragment of DNA of greater than 100 kb was recovered from EPI300 cells harboring the plasmid as can be seen by running supercoiled plasmid DNA on an agarose gel (FIG. 5).

Sequencing of the plasmid (ILLUMINA® MISEQ®) confirmed the expected sequence, demonstrating that pVnoriCII can be used to clone fragments of exogenous DNA of greater than 100 kb in E. coli.

Improved E. coli/V. Natriegens Shuttle Vector:

In order to improve upon the design of pVnatoriCII, a second V. natriegens ChII plasmid was created (designated pVnatCII-YACTRP-copycontrol (SEQ ID NO: 4)) by leveraging features from the following plasmids:

From pVnoriCII:

-   -   oriT for RP4-mediated conjugal transfer     -   V. natriegens chill origin of replication for low copy         replication of plasmid

From pCC1BAC™ from EPICENTRE®:

-   -   oriV for use with E. coli strains containing the trfA gene         product under an inducible promoter (e.g., EPI300 cells from         Epicentre®)     -   chloramphenicol resistance marker for antibiotic selection     -   Multiple Cloning Site (MCS) with convenient restriction enzyme         cut sites     -   loxP site for recombination via Cre-recombinase

In addition, the plasmid also contains:

-   -   ARS/CEN for stabile replication in Saccharomyces cerevisiae     -   Trp gene for selection in a Trp auxotroph of Saccharomyces         cereviciae

The vector map and sequence of pVnatCII-YACTRP-copy control are shown in FIG. 6. The plasmid pVnatCII-YACTRP-copycontrol was replicated in E. coli at low copy, and the copy number was also increased by supplementing the trfA gene product in trans (e.g., via EPI300 cells, which contain an inducible copy of tifA on the chromosome).

The plasmid pVnatCII-YACTRP-copycontrol was also introduced into E. coli, V. natriegens, and Saccharomyces cerevisiae via transformation and maintained under the appropriate selection.

Example 7 Use of a Suicide Plasmid System to Engineer the Genome of V. natriegens

We have developed a suicide plasmid system that can be used to remove endogenous DNA sequence or introduce exogenous DNA into the chromosome of V. natriegens. The plasmid was constructed with the following DNA elements in the following order:

a) 500 bp of chromosomal sequence directly upstream of the location where an insertion/deletion event was desired to start.

b) A “knock-out/knock-in” cassette containing an antibiotic resistance marker (e.g., Cm antibiotic resistance marker (from pACYC184)) flanked by lox66 and lox71 sites. In addition, if exogenous DNA was to be added into the chromosome, that DNA was contained in this fragment after the lox-bounded Cm marker.

c) 500 bp of chromosomal sequence directly downstream of the location where an insertion/deletion event was desired to end.

d) the R6K origin of replication from pR6Kan (Epicentre®).

e) the RK2 origin of transfer (oriT) from pRL443.

f) the ccdB toxin under control of the arabinose-inducible araBAD promoter (SEQ ID NO:5) (the araC gene and araBAD promoter are from plasmid pKD46).

Because the plasmid lacks the π replication protein necessary to initiate replication from the R6K origin, the plasmid will only replicate when π is supplied in trans (e.g., from the EC100D pir-116 strain from Epicentre®). The plasmid was introduced into an E. coli strain capable of supplying the π protein in trans that also contained the conjugation machinery from plasmid RP4 (we use strain S17-12\pir). The strain was then mated with V. natriegens (following the conjugation protocol described in Example 2) to allow mobilization of the plasmid from the donor E. coli strain to V. natriegens. Because the plasmid is incapable of replicating in V. natriegens, the only way that antibiotic-resistant clones were isolated was if the plasmid integrated into the chromosome via the regions of the plasmid that are homologous to the V. natriegens genome. Double-crossover integration events were selected for by growing the strain in media (e.g., LB) containing 0.2-0.4% L-arabinose as well as an antibiotic (the antibiotic which is contained in the cassette flanked by homology to the V. natriegens genome). The presence of arabinose induced the araBAD promoter, thereby producing the ccdB toxin and removing cells that had not undergone integration via double-crossover recombination from the population (the toxin is not present in cells that have undergone double-crossover recombination). Surviving clones were screened for successful integration via standard methods.

Use of the System to Remove Endogenous DNA Sequence from the Chromosome

In this embodiment, the “knock-out/knock-in” cassette was composed simply of an antibiotic resistance gene flanked by lox sites oriented in the same direction (e.g., the orthogonal and uni-directional lox66/lox71 pair). The “knock-out/knock-in” cassette was flanked on either side by 500-750 bp of V. natriegens chromosomal sequence that was chosen such that the antibiotic cassette was flanked by 500-750 bp of sequence immediately upstream of the start point of the desired deletion, and 500-750 bp immediately downstream of the end point for the desired deletion. Upon successful integration via double-crossover recombination, the region of the genome to be deleted was replaced by the antibiotic cassette flanked by lox sites. The antibiotic cassette was later removed from the genome via the expression of Cre recombinase, which recombined the lox sites, thus looping the antibiotic cassette out of the chromosome (see discussion of engineering with Cre recombinase in Example 8). In some examples, we used this technique to remove the ORF for the Dns exonuclease. In another example, we used this technique to remove a 28 kb region of Chromosome I from strain CCUG 16374 harboring a putative restriction-modification system (FIG. 7).

Use of the System to Introduce Exogenous DNA Into the Chromosome

In this embodiment, the “knock-out/knock-in” cassette was composed of an antibiotic resistance gene (which may or may not be flanked by lox sites oriented in the same direction) as well as additional exogenous DNA to be added into the chromosome. The “knock-out/knock-in” cassette was flanked on either side by 500 bp of V. natriegens chromosomal sequence that was chosen such that the “knock-in” cassette is flanked by 500 bp of sequence immediately upstream of the start point of the desired insertion, and 500 bp immediately downstream of the end point for the desired insertion. Upon successful integration via double-crossover recombination, the exogenous DNA along with the antibiotic marker was inserted into the genome at the desired location. If the antibiotic cassette is flanked by lox sites, it was later removed from the genome via the expression of Cre recombinase, which recombined the lox sites, looping the antibiotic cassette out of the chromosome (see discussion of engineering with Cre recombinase in Example 8). In some examples, we used this technique to introduce an inducible T7 RNA polymerase gene (SEQ ID NO:7) into the genome (see discussion of protein expression via an inducible T7 RNA polymerase in Example 9).

Example 8 Use of Site-Specific Recombinases to Engineer the Genome of V. natriegens

The use of site specific recombinases along with their target sequences was used to carry out insertions and deletions in the chromosome of V. natriegens and could additionally be used to carry out inversions. We have demonstrated the use of the Cre-lox system to remove sequences present in the chromosome that have been flanked by lox sites.

In Example 7 (Use of a suicide plasmid system to engineer the genome of V. natriegens), a chloramphenicol marker flanked by lox66 and lox71 sites (that are oriented in the same direction) was introduced into the chromosome in such a manner as to replace the entire ORF for the Dns nuclease. By expressing Cre recombinase, recombination between the lox sites resulted in the removal of the antibiotic marker from the chromosome, leaving behind a native loxP site (thus allowing us to recycle our antibiotic marker). To this end we designed the plasmid pACYCtetoriTCre, which contains:

-   -   a) the p15a origin of replication from plasmid pACYC184     -   b) the tetracycline resistance cassette from plasmid pJB3Tc20     -   c) the RK2 the RK2 origin of transfer (oriT) from pRL443     -   d) the temperature-inducible Cre expression cassette from         plasmid 705-Cre (from Gene Bridges GmbH)

Introduction of the plasmid into the strain (carrying the lox site flanked modification) via electroporation, followed by incubation at 37° C. (to induce expression of Cre recombinase) resulted in the desired phenotype (i.e., a strain that had undergone Cre-mediated recombination to remove the antibiotic marker).

In addition to carrying out deletions, this system can be used to introduce novel DNA into a chromosome (via recombination with an exogenous circular DNA containing a lox site) and additionally or alternatively to invert regions of the chromosome (with proper orientation of the lox site).

Analogous systems are envisioned which rely on other site-specific recombinases or integrases (e.g., phiC31 integrase, bxb1 integrase, etc.).

Example 9 The Introduction of an Inducible T7 RNA Polymerase Gene into the V. natriegens Chromosome and its Use in Recombinant Protein Expression

Using the suicide plasmid system described in Example 7, we introduced the gene for T7 RNA polymerase (SEQ ID NO:7) under the control of either a) The arabinose-inducible araBAD promoter (SEQ ID NO:5) and araC regulator protein (from E. coli); orb) the IPTG-inducible lac operon regulatory elements and lad regulator protein (SEQ ID NO:6) (from E. coli) into the chromosome of V. natriegens. This system allowed for inducible, robust protein expression from a plasmid-borne gene under control of the T7 promoter.

In conjunction with the strain, we designed a plasmid known as pET325Cm-YGFP which is based off of plasmid pET28a (Novagen) and contains the YGFP fluorescent protein under the T7 promoter.

The plasmid was introduced into V. natriegens araBAD-T7 and lacI-T7 strains as well as the wild type (wt) parental strain via electroporation, as described above (Example 3). Strains harboring the plasmid were grown up overnight in Brain Heart Infusion Broth+v2 salts with 12.5 ug/mL chloraphenicol at 30° C. with shaking at 200 RPM (v2 salts means the media was supplemented with additional salts at the following concentrations: 204 mM NaCl, 4.2 mM KCl, 23.14 mM MgCl₂). The next day 50 mL of either LB+v2 salt media or BHI+v2 salt media with 15 ug/mL chloramphenicol in a 250 mL baffled flask was inoculated with 1/100^(th) volume of overnight culture and incubated at 30° C. When the OD600 was between 0.6 and 0.9 the cultures were induced with 1 mM IPTG (wt and lacI-t7 strains) or 1 mM IPTG+0.2% arabinose (araBAD-T7 strain). At 6.5 hrs post induction, the cultures were retrieved and the cells were harvested via centrifugation. The pellets were suspended in buffer (50 mM Tris pH 7.4, 300 mM NaCl, 5 mM imidazole) to a total volume of about 7 mL. The cells were then imaged under white light (FIG. 8A), or under a blue light transilluminator with orange filter (FIG. 8B). As can be seen in FIG. 8, the wt strain, even when induced did not make any protein. Both versions of the T7 expression system (araBAD-T7 and lacI-T7) expressed YGFP. The pellets were lysed via sonication, clarified via centrifugation, and the lysate was analyzed by SDS-PAGE (FIG. 9). The overexpressed YGFP construct is visible in the lacI-T7 and araBAD-T7 strains.

Many configurations of this system are envisioned. In some embodiments the RNA polymerase could reside on a plasmid and the gene that is to be overexpressed could be cloned into any number of vectors under control of the T7 promoter.

Analogous expression strains could be generated using other configurations of chromosomally integrated or plasmid-borne inducible RNA polymerases, relying on other RNA polymerases (e.g., SP6 RNA polymerase, etc.) or inducible promoters (e.g., other chemically inducible promoters, temperature inducible promoters, etc.)

Example 10 Chemically Competent V. natriegens Cells

This example provides a protocol for preparing chemically competent V. natriegens cells. This protocol has been used to prepare competent cells of CCUG 16364 and ATCC 14048 strains (that have a deletion of the Dns chromosomal nuclease) that achieve transformation efficiencies between 10⁵ and 10⁶ cfu/ug DNA (plasmid pACYC184).

Preparation of chemically competent cells. 10 mL of BHI+v2 salts (Brain Heart Infusion Broth supplemented with salts (204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl₂)) is inoculated with a colony of V. natriegens (carrying a deletion of the chromosomal Dns endonuclease) from an agar plate, and incubated overnight at 30° C. with agitation at 200 RPM. On the following day, 150 mL of the same growth media is inoculated with the overnight culture at a dilution of 1:100 (overnight culture:fresh media). The culture is grown at 30° C. in a baffled flask with shaking at 200 RPM until an OD500 of 0.4. Working as quickly as possible, all subsequent steps are performed at room temperature. The culture is split into four 50 mL centrifuge tubes, and the cells are pelleted by centrifuging at 2300×g for 7 mM The supernatant is carefully removed, and each pellet is gently suspended with 5 mL of 100 mM MgCl₂. The cells from all four centrifuge tubes are consolidated into two centrifuge tubes, the volume of each is brought up to 30 mL with 100 mM MgCl₂, and the tubes are mixed by gentle inversion. Cells are pelleted by centrifuging at 2300×g for 4.5 mM The supernatant is completely removed from each tube, and the pellets are each suspended in 5 mL of 100 mM CaCl₂. The two suspensions are combined, and the volume adjusted to 30 mL with 100 mM CaCl₂. The tubes are gently mixed by inversion and then incubated at room temperature for 20 mM Following the incubation, cells are pelleted by centrifuging at 2300×g for 4.5 mM The supernatant is drawn off, each the pellets are collected in a combined volume of ˜1 mL of 100 mM CaCl₂, transferred to a 1.5 mL Eppendorf tube, and pelleted by centrifuging at 2300×g for 1-2 mM The supernatant is removed, and the cells are resuspended in resuspension buffer (a modified version of that described by Inoue¹: 55 mM MnCl₂, 15 mM CaCl₂, 250 mM KCl, 10 mM PIPES (from 0.5 M pH 6.7 stock), 10% glycerol (w/v), 5% PEG 8000). Cells are aliquoted into pre-chilled tubes, frozen in a dry ice bath, and are stored at −80° C. until use.

Transformation of chemically competent cells. A vial of competent cells is retrieved from storage at −80° C. and allowed to thaw on ice. Plasmid DNA and 50 μL of competent cells are combined and gently mixed in a pre-chilled 1.5 mL microfuge tube and incubated on ice for 20-30 mM During incubation, a tube for each transformation is prepared by adding 1 mL BHI+v2 salts (Brain Heart Infusion Broth supplemented with salts (204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl₂)) to a culture tube and warming to 37° C. in a water bath. At the end of the ice incubation, use some of the pre-warmed media to transfer the cells out of the Eppendorf tube and into the culture tube. Let tube sit at 37° C. for 1 mM Following 1 minute at 37° C., allow the cells to recover by incubating at 30° C. for 2 hrs. with agitation at 200 RPM. Plate out dilutions of cells on pre-warmed selective plates and incubate overnight at 30° C.

Example 11 Engineering Cold Tolerance into V. natriegens Via Inclusion of Heterologous Genes

Storage of V. natriegens on solid media (LB agar) is not robust. Colonies on LB agar plates can maintain viability for at least 3 weeks at room temperature, but colonies on plates stored at 4° C. were not viable after 3 weeks (FIG. 14A).

This Example illustrates that the cold tolerance of V. natriegens was improved by importing a heterologous ROS detoxification system from other organisms. V. natriegens strains carrying various ROS detoxification systems remained viable and culturable after exposure to low temperatures. V. natriegens having the E. coli alkyl hydroperoxide reductase operon (ahpCF), (katG or katE) (FIG. 14C), catalase (FIG. 14B), or glutathione S-transferase (gstA) genes (under control of heterologous promoters) on plasmids showed a marked improvement of viability over the wt cells after 11 days of cold storage, with cells carrying katG showing robust survival out to at least 3 weeks (ahpCF, katG, and katE results pictured in FIG. 14C). The inclusion of these heterologous genes on plasmids or integrated into the V. natriegens chromosome was therefore shown to preserve and extend the viability and culturability of cells exposed to refrigeration.

Example 12 Sucrose Induced Protein Secretion in V. natriegens

This Example shows the ability of a protein production host to secrete the synthesized protein directly into the growth media when induced with sucrose. V. natriegens strains were grown in a minimal media with a variety of different carbon sources. A minimal growth media was prepared by combining 66 mL 10× phosphate/citric acid buffer (133 g/L KH₂PO₄, 40 g/L (NH₄)₂HPO₄, 17 g/L citric acid, pH 6.3), 27.9 mL 70% glucose, 1.58 mL MgSO₄●7H₂O (500 g/L stock), 45.6 mL 5 M NaCl, and 518.92 mL ddH₂O. The pH was adjusted to 6.8 and the media filter sterilized. 10 ml aliquots of 10 mL of BHI+v2 salts (Brain Heart Infusion Broth supplemented with salts (204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl₂)) were inoculated with either V. natriegens ATCC 14048 or CCUG 16374 from glycerol stocks and grown overnight at 30° C. The next day, 625 μL of each strain was used to inoculate three separate 50 ml centrifuge tubes containing 12.5 ml of the aforementioned minimal growth media. Of the three tubes for each strain, one was left as is, the second was supplemented with 0.2% (w/v) additional glucose, and the third was supplemented with 0.2% (w/v) sucrose. The three cultures for each strain were grown at 37° C. in a shaking incubator for 5 hours and 20 minutes. Cultures were spun down at 3000 rpm for 3 min at room temperature, and the supernatant was carefully drawn off and filtered through a 50 ml 0.22 uM Tube Top filter (430320). The flow-through (about 10 ml) was transferred to a 15 ml centrifuge tube. To this, 2.5 ml of 100% TCA (Trichloroacetic acid) was added, mixed by inversion, and incubated on ice for 10 min. The mixture was spun down at 7197×g at room temperature for 10 min. The resulting pellet was washed twice with 500 μl acetone, also spun at max speed for 10 min. At this point, samples were either analyzed by SDS-PAGE or submitted for 2D nano LC-MS/MS analysis.

For samples analyzed by SDS-PAGE, the tubes were placed in an 85° C. heat block for 5-10 min to remove acetone. Each pellet was then resuspended with 10 ul 4×LDS BOLT® Sample Buffer, 4 μl 10× BOLT® Sample Reducing agent and 26 μL ddH₂O by pipetting up and down vigorously. Samples were heated at 85° C. for about 7 min, spun down briefly, and loaded onto BOLT® 4-12% Bis Tris Plus 10-well protein gel with MES SDS running buffer, and run at 180 V for approximately 30 min Gels were washed and stained with Coomasie G-250 and destained O/N. Interestingly, culturing in the presence of sucrose resulted in the presence of several proteins in high concentration in the growth media (FIG. 13).

Acetone-washed protein precipitates were subjected to 2D nano LC-MS/MS analysis. Raw MS/MS spectra were searched against a protein database for the strain prepared from the genome annotation as well as a decoy sequence database, giving a false discovery rate of <<0.1% at the peptide level. Proteomics analysis enabled positive identification of the V. natriegens secretome (FIG. 12). The most abundant protein identified by proteomics in the sucrose-containing media is consistent with the molecular weight of the protein identified via SDS-PAGE, and was annotated as an arabinase/levansucrase/invertase, a class of proteins known to be involved in sucrose catabolism. MS analysis revealed that the putative tag has been removed from the secreted protein during the export process. In view of these data it is seen that one can achieve recombinant protein expression by fusing these secretion tags with a recombinant protein of interest in order to direct the protein into the growth media.

Example 13 Effect of Removal of Endonuclease Dns from V. natriegens CCUG 16374

The cellular endonuclease (Dns) in V. natriegens CCUG 16374 was identified and removed and found to markedly improve the quality and stability of isolated plasmid DNA. Plasmid DNA isolated from V. natriegens CCUG 16374 with or without the endogenous Dns nuclease is depicted in FIG. 14. Plasmid pACYC184 was isolated from CCUG 16374 with or without a chromosomal deletion of Dns and purified. The isolated DNA was then subjected to restriction digest with NheI and analyzed by gel electrophoresis. It was found that in the nuclease deficient strain, the integrity of the DNA was greatly preserved (FIG. 14).

Although the disclosure has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the disclosure. Accordingly, the disclosure is limited only by the following claims.

Example 14 Secretion of Modified Levansucrase

The signal sequence of SEQ ID NO: 8 was fused to modified forms of the endogenous V. natriegens levansucrase as three recombinant constructs. All three constructs were efficiently secreted by V. natriegens, as evidenced by FIG. 16, which depicts an SDS-PAGE gel of extracellular culture supernatant.

Example 15 Periplasmic Secretion of Beta-Lactamase in V. natriegens

The V. natriegens trimethylamine-N-oxide signal sequence of SEQ ID NO: 28 was fused to the N-terminus of a variant of beta-lactamase lacking its natural N-terminal signal sequence. The construct was inserted into a pMB1 plasmid where the recombinant gene is under a lac promoter. The plasmid was introduced and conferred resistance to carbenicillin as determined by growth on LB agar plates containing carbenicillin.

Beta-lactamase confers resistance to carbenicillin only if it is successfully secreted into the periplasm of gram negative bacteria, and depends upon its natural N-terminal signal sequence for secretion. Cells carrying the recombinant plasmid described above were resistant to carbenicillin (50 ug/mL), demonstrating that the cells were successfully secreting beta-lactamase since Vibrio sp. does not naturally have beta lactamase.

Although the disclosure has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the disclosure. Accordingly, the disclosure is limited only by the following claims. 

The invention claimed is:
 1. A cold tolerant Vibrio natriegens organism comprising an exogenous sequence encoding one or more enzymes from a reactive oxygen species detoxification system and able to generate new colonies after being stored at a temperature of 4° C. or less for at least 14 days.
 2. The organism of claim 1 wherein the organism comprises a heterologous nucleotide sequence encoding any one or more of: a) one or more alkyl hydroperoxide reductase gene(s) under the control of an exogenous promoter and operable in Vibrio natriegens, or b) a catalase gene under the control of an exogenous promoter and operable in Vibrio natriegens, or c) a glutathione S-transferase gene under the control of an exogenous promoter and operable in Vibrio natriegens.
 3. The organism of claim 2 wherein the heterologous nucleotide sequence encodes an alkyl hydroperoxide reductase operon under the control of an exogenous promoter and operable in Vibrio natriegens.
 4. The organism of claim 3 wherein the alkyl hydroperoxide reductase operon is the alkyl hydroperoxide reductase operon from E. coli (ahpCF).
 5. The organism of claim 2 wherein the heterologous nucleotide sequence encodes a catalase gene under the control of an exogenous promoter and operable in Vibrio natriegens.
 6. The organism of claim 3 wherein the catalase gene is katG or katE under the control of a heterologous promoter and operable in Vibrio natriegens.
 7. The organism of claim 2 wherein the heterologous nucleotide sequence encodes a glutathione S-transferase gene under the control of an exogenous promoter and operable in Vibrio natriegens.
 8. The organism of claim 2 wherein the any one or more of alkyl hydroperoxide reductase operon, the catalase gene, and the glutathione S-transferase gene are comprised on ChI or ChII.
 9. The organism of claim 2 further comprising an exogenous vector, and wherein the alkyl hydroperoxide reductase operon, or the catalase gene, or the glutathione S-transferase gene are comprised on the vector.
 10. The organism of claim 2 comprised on a solid media.
 11. The organism of claim 2 wherein the heterologous nucleotide sequence encoding any one of a), b), or c) enables the organism to generate new colonies after storage at about 4° C. for at least 19 days. 